Production of pathogen resistant plants

ABSTRACT

Methods for increasing resistance in plants to pathogens by the expression of a hydrogen peroxide/reactive oxygen species producing enzyme or an oxalate degrading enzyme. The present invention relates to a method of producing a pathogen resistant hybrid plant by crossing the appropriate transgenic expressing a hydrogen peroxide/reactive oxygen species producing enzyme or an oxalate degrading enzyme with pathogen tolerant lines or inbreds obtained through conventional genetic manipulations, or by transformation of tolerant plants or plant tissues with a hydrogen peroxide/reactive oxygen species producing gene or by altering the expression of an endogenase hydrogen peroxide/reactive oxygen species producing gene. The synergistic effect of expression of a hydrogen peroxide/reactive oxygen species producing enzyme or an oxalate degrading enzyme in a tolerant background gives significant and unexpectedly high resistance to the pathogens.

CROSS REFERENCE PARAGRAPH

This Application is a division of U.S. Ser. No. 09/115,488, now U.S.Pat. No. 6,166,291 filed Jul. 14, 1998, and claims the benefit of U.S.Provisional Application No. 60/053,125, filed Jul. 18, 1997 and areherein incorporated by reference.

TECHNICAL FILED

This invention relates to the genetic improvement of plants by the useof recombinant DNA techniques. Particularly, but not exclusively, theinvention relates to the improvement of the tolerance of plants topathogen attack.

BACKGROUND

Diseases of plants have caused an ongoing and constant problem in plantcultivation. The fungal pathogen, Sclerotinia sclerotiorum, inparticular is said to cause disease in nearly 400 plant species.Sclerotinia sclerotiorum appears to be among the most nonspecific,omnivorous, and successful of plant pathogens. (Purdy, L. H.,Phytopathology 69: 875-880 (1979))

Sclerotinia infections in sunflower, for example, are considered themajor disease problems of the crop yet little genetic resistance iscurrently available to breeding programs to combat the various forms ofthis fungal infection. In fact, there are no major gene resistancemechanisms that have been defined in any species affected by thispathogen.

Oxalate (oxalic acid) is a diffusable toxin associated with variousplant diseases, particularly those caused by fungi. While some leafygreen vegetables, including spinach and rhubarb, produce oxalate as anutritional stress factor, certain pathogens synthesize and export largeamounts of oxalate to assist in the establishment and spread of theorganism throughout infected hosts. Oxalate is used by pathogens to gainaccess into and subsequently throughout an infected plant. See forexample, Mehta and Datta, J. Biol. Chem., 266: 23548-23553, andpublished PCT Application WO 92/14824 published in Sep. 3, 1992. Fieldcrops such as sunflower, bean, canola, alfalfa, soybean, flax,safflower, peanut, clover, maize, sorghum, wheat, rice, as well asnumerous vegetable crops, flowers, and trees are susceptible tooxalate-secreting pathogens. For example, fungal species including, butnot limited to, Sclerotinia, Sclerotium, Aspergillus, Streptomyces,Penicillium, Pythium, Pacillus, Mycena, Leucostoma, Rhizoctonia andSchizophyllum use oxalic acid to provide an opportunistic route of entryinto plants, causing serious damage to crops such as sunflower.

Enzymes that utilize oxalate as a substrate have been identified. Theseinclude oxalate oxidase (wheat oxalate oxidase is sometimes calledgermin) and oxalate decarboxylase. Oxalate oxidase catalyzes theconversion of oxalate to carbon dioxide and hydrogen peroxide. A geneencoding barley oxalate oxidase has been cloned from a barley root cDNAlibrary and sequenced (See: PCT publication No. WO 92/14824, publishedin Sep. 3, 1992). A gene encoding wheat oxalate oxidase activity hasbeen isolated and sequenced, and the gene has been introduced into acanola variety (PCT publication No. WO 92/15685 published in Sep. 3,1992, Drawtewka-Kos, et al., J. Biol. Chem., 264 (9): 4896-4900 (1991)).Oxalate decarboxylase converts oxalate to carbon dioxide and formicacid. A gene encoding oxalate decarboxylase has been isolated fromCollybia velutipes (now termed Flammulina velutipes) and the cDNA clonehas been sequenced (WO 94/12622, published in Jun. 9, 1994). Inaddition, another oxalate decarboxylase gene has been isolated fromAspergillus phoenices (U. S. patent application No. 08/821,827, now U.S.Pat. No. 6,297,425 filed on March 21, 1997).

Another gene which does not degrade oxalate, but which has been shown tohelp in the control of plant fungal pathogens is glucose oxidase. (SeeU.S. Pat. No. 5,516,671, filed on Nov. 3, 1994 and Wu, et al., PlantCell, 7: 1357-1368 (1995)). In the presence of oxygen, glucose oxidasecatalyzes the oxidation of glucose to ∂-gluconolactone and hydrogenperoxide. It is thought that the hydrogen peroxide and the∂-gluconolactone, which is known as glycosyltransferase inhibitor, areresponsible for the anti-pathogenic mode of action.

In many plants, attempted infection by avirulent pathogens triggers theactivation of multiple defenses that may be accompanied by ahypersensitive response (HR) or collapse of host tissue around the siteof pathogen penetration. A consequence of these responses is arestriction of pathogen spread within the host and frequentlydevelopment of systemic acquired resistance (SAR) to subsequentinfection by pathogens that may be taxonomically distant to the initialpathogen. For e.g., SAR induced by virus inoculation may be effectiveagainst subsequent attack by bacterial or fungal pathogens or viceversa. One of the earliest responses of the plant to infection is anoxidative burst which can be detected as an increased accumulation ofsuperoxide (O₂) and/or hydrogen peroxide (H₂O₂). O₂ is very reactive andcan form other reactive oxygen species, including hydroxyl radical (OH)and the more stable H₂O₂. H₂O₂ accumulation may trigger enhancedresistance responses in a number or ways: 1. Direct antimicrobialactivity, 2. Act as a substrate for peroxidases associated with ligninpolymerization and hence cell wall strengthening, 3. Via still to bedetermined mechanisms act as a signal for activation of expression ofdefense related genes, including those that result in stimulation ofsalicylic acid (SA) accumulation. SA is thought to act as an endogenoussignal molecule that triggers expression of genes coding for severalclasses of pathogenesis-related proteins (PR proteins). Some of the PRproteins have antimicrobial enzymatic activities, such as glucanases andchitinases. The function of other PR proteins in defense still needs tobe elucidated. Moreover, SA may potentiate the oxidative burst and thusact in a feedback loop enhancing its own synthesis. SA may also beinvolved in hypersensitive cell death by acting as an inhibitor ofcatalase, an enzyme that removes H₂O₂. 4. H₂O₂ may trigger production ofadditional defense compounds such as phytoalexins, antimicrobial lowmolecular weight compounds. For a review on the role of the oxidativeburst and SA please see Lamb, C. and Dixon, R. A., Ann. Rev. Physiol.Plant Mol. Biol., 48: 251-275 (1997). A high level of salicylic acid isassociated with disease lesion mimic symptoms. Thus, the oxidative burstis the initial signal of a pathogen's attack, but one that is notpermitted to be maintained by the plant. Even plants that are able tomount a defense are usually not immune to the disease. The pathogen isoften able to inflict significant damage, although the plant may not diefrom the disease. Plants stressed because of pathogen damage are lesslikely to yield well and are often more susceptible to other types ofpests.

In the present invention, it is demonstrated that the transgene encodinghydrogen peroxide/reactive oxygen species producing enzyme or an oxalatedegrading enzyme is able to confer a significant pathogen resistanceresponse in sunflower, canola, and soybean. Further, pathogen resistantsunflower expressing oxalate oxidase induces the expression ofpathogenesis-related genes resulting in the accumulation of high levelsof PR-1, chitinase and glucanase PR proteins as well as highly elevatedlevels of salicylic acid. Induction of the host defense systems has beenshown in numerous cases to cause broad spectrum resistance to pathogens.For example, Chen, et al. in a 1993 Science article discusses thatinfection of plants by a pathogen often leads to enhanced resistance tosubsequent attacks by the same or even unrelated pathogens (Chen, etal., Science, 262: 1883-1886 (1993)).

A lesion mimic-like phenotype also is observed in these SMF-3 transgenicplants. Sclerotinia resistant F1 hybrids of oxalate oxidase or oxalatedecarboxylase transgenic plants crossed with existing Sclerotiniatolerant sunflower lines generated near-immune plants with no lesionmimic symptoms. A similar near-immune phenomenon is also observed withF1 hybrids of canola oxalate oxidase transgenics and an existingSclerotinia tolerant line. Thus the synergistic effect of a hydrogenperoxide/reactive oxygen species producing enzyme or an oxalatedegrading enzyme in a plant with a genetic pathogen tolerance gives riseto a near immune plant. A Sclerotinia immune plant has never beendescribed before. It is now possible to take tolerant plants and makethem immune or nearly immune to Sclerotinia. A pathogen immune plant canbe expected to survive and yield well under pathogen challenge withoutthe need for externally applied control agents such as chemicalfungicides. Therefore, producers are spared the expense and effortrequired to treat fields for disease problems. In the case ofSclerotinia, for example, current treatment protocols are only partiallyeffective and cost prohibitive. An effective transgenic approach toSclerotinia disease control would therefore be of significant utility.

SUMMARY

The present invention relates to a method of producing resistance inplants to pathogens by the expression of a hydrogen peroxide/reactiveoxygen species producing enzyme such as, but not limited to, oxalateoxidase (such enzyme(s) generally referred to herein as “reactive oxygenproducing enzyme(s)”) or a oxalate degrading enzyme such as, but notlimited to oxalate decarboxylase in a conventionally tolerantbackground. The present invention also relates to a method of producinga Sclerotinia near-immune hybrid plant by crossing the appropriatetransgenic plant expressing a hydrogen peroxide/reactive oxygen speciesproducing enzyme or an oxalate degrading enzyme with pathogen tolerantlines or inbreds obtained through conventional genetic manipulations.Crossing the transgenic plant into a pathogen tolerant backgroundproduces a resistant plant with a high level of pathogen resistance, andno disease lesion mimic symptoms. The synergistic effect of expressionof a hydrogen peroxide/reactive oxygen species producing enzyme or anoxalate degrading enzyme in a tolerant background gives significant andunexpectedly high resistance to the pathogen Sclerotinia.

Alternatively, the tolerant background plant could be used fortransformation resulting in direct integration of a gene encoding thehydrogen peroxide/reactive oxygen species producing enzyme or oxalatedegrading enzyme in a tolerant background.

Another embodiment of the invention relates to the overexpression of anendogenous plant gene. In some embodiments, isolated nucleic acids thatserve as promoter or enhancer elements can be introduced in theappropriate position (generally upstream) of an endogenous form of thegene(s) encoding an enzyme of the present invention so as to up or downregulate expression of that enzyme.

Plants that could be transformed and made disease resistant include, butin no way are limited to; sunflower, bean, canola, alfalfa, soybean,flax, safflower, peanut, clover, maize, sorghum, wheat, rice, as well asnumerous vegetable crops, flowers, and trees.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the slope of the oxalate oxidase activity versus the numberof sclerotia in sunflower.

FIG. 2 shows the slope of the oxalate oxidase activity versus thesclerotia weight in sunflower.

FIG. 3 shows the frequency by range of sclerotia weights in oxalateoxidase expression sunflower plants versus non-oxalate oxidaseexpression sunflower plants.

FIG. 4 shows the oxalate oxidase activity versus the sclerotia weight insunflower plants.

FIG. 5 is a graph of sclerotia weight versus PR protein expression insunflower plants.

FIG. 6 is a graph of the slope of the oxidase activity versus the PRprotein rating in sunflower plants.

FIG. 7 is a graph of the slope of the oxalate decarboxylase enzymeactivity versus the sclerotia weight in sunflower plants.

FIG. 8 is a graph of the oxalate decarboxylase activity range versus PRrating.

FIG. 9 shows the slope of the oxalate oxidase activity versus the fungallesion rating.

FIG. 10 shows sclerotial body weight comparisons of various sunflowervarieties. Sclerotinia sclerotiorum inoculated sunflower lines wereallowed to complete their life cycle and then harvested in order tocollect and measure sclerotial bodies. The resistance or susceptibilityand presence or absence of the transgene are indicated by the differentbars and numbers. The number labeling each bar corresponds to thespecific genotype and whether or not it is trangenic according to Table4.

FIG. 11 shows the effect of sunflower line PK68F and the expression ofoxalate oxidase on Sclerotinia sclerotiorum sclerotial body weight andnumber. Data is presented which separates the lines into appropriatecomparisons; each of the non-transgenic parent lines, the non-transgenichybrids (PK68F/SMF3, PK93M/SMF3, PR118M/SMF3 and PR126M/SMF3),transgenic SMF3 event 193870 (SMF3/35Soxox), and the transgenic hybridwith SMF3 event 193870 (PK68F/35Soxox, PK93M/35Soxox, PR118M/35Soxox,and PR126M/35Soxox). Numerical identification, listed under the bars ineach graph, correspond to those presented in the table associated withTable 4. (abbreviations: wt.—weight, ct.—count, oxox=oxalate oxidase,35S=35S promoter)

FIG. 12 shows the effect of sunflower line PK93M and the expression ofoxalate oxidase on Sclerotinia sclerotiorum sclerotial body weight andnumber. Data is presented which separates the lines into appropriatecomparisons; each of the non-transgenic parent lines, the non-transgenichybrids (PK68F/SMF3, PK93M/SMF3, PR118M/SMF3 and PR126M/SMF3),transgenic SMF3 event 193870 (SMF3/35Soxox), and the transgenic hybridwith SMF3 event 193870 (PK68F/35Soxox, PK93M/35Soxox, PR118M/35Soxox,and PR126M/35Soxox). Numerical identification, listed under the bars ineach graph, correspond to those presented in the table associated withTable 4. (abbreviations: wt.—weight, ct.—count, oxox=oxalate oxidase,35S=35S promoter)

FIG. 13 shows the effect of sunflower line PR 118M and the expression ofoxalate oxidase on Sclerotinia sclerotiorum sclerotial body weight andnumber. Data is presented which separates the lines into appropriatecomparisons; each of the non-transgenic parent lines, the non-transgenichybrids (PK68F/SMF3, PK93M/SMF3, PR118M/SMF3 and PR126M/SMF3),transgenic SMF3 event 193870 (SMF3/35Soxox), and the transgenic hybridwith SMF3 event 193870 (PK68F/35Soxox, PK93M/35Soxox, PR118M/35Soxox,and PR126M/35Soxox). Numerical identification, listed under the bars ineach graph, correspond to those presented in the table associated withTable 4. (abbreviations: wt.—weight, ct.—count, oxox=oxalate oxidase,35S=35S promoter)

FIG. 14 shows the effect of sunflower line PR126M and the expression ofoxalate oxidase on Sclerotinia sclerotiorum sclerotial body weight andnumber. Data is presented which separates the lines into appropriatecomparisons; each of the non-transgenic parent lines, the non-transgenichybrids (PK68F/SMF3, PK93M/SMF3, PR118M/SMF3 and PR126M/SMF3),transgenic SMF3 event 193870 (SMF3/35Soxox), and the transgenic hybridwith SMF3 event 193870 (PK68F/35Soxox, PK93M/35Soxox, PR118M/35Soxox,and PR126M/35Soxox). Numerical identification, listed under the bars ineach graph, correspond to those presented in the table associated withTable 4. (abbreviations: wt.—weight, ct.—count, oxox=oxalate oxidase,35S=35S promoter)

FIG. 15 shows the detection of galactose oxidase in maize callus.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

A structural gene is a region of DNA having a sequence that istranscribed into messenger RNA (mRNA) that is then translated into asequence of amino acids characteristic of a specific polypeptide.Structural genes also include gene encoding RNA products directly suchas genes encoding transfer RNA (tRNA).

As used herein promoter includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A plantpromoter is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells such Agrobacterium or Rhizobium.Examples are promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, seeds, fibres, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to, as tissuepreferred. A cell type specific promoter primarily drives expression incertain cell types in one or more organs, for example, vascular cells inroots or leaves. An inducible promoter is a promoter that is underenvironmental control. Examples of environmental conditions that mayeffect transcription by inducible promoters include anaerobic conditionsor the presence of light. Another type of promoter is a developmentallyregulated promoter, for example a promoter that drives expression duringpollen development. Tissue preferred, cell type specific,developmentally regulated, and inducible promoters constitute the classof non-constitutive promoters. A constitutive promoter is a promoterthat is active under most environmental conditions.

An element is a region of DNA having a sequence that is involved in theregulation of gene expression. Examples of elements include terminators,introns, polyadenylation sequences, nucleic acid sequences encodingsignal peptides which permit localization within a plant cell orsecretion of the protein from the cell, or as in the present invention anucleic acid sequence that regulates transcription in response to aninducer or the signal produced in response to an inducer.

An enhancer is a DNA regulatory region that can increase the efficiencyof transcription, and may or may not be independent of the distance ororientation of the enhancer relative to the start site of transcription.

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into protein.

A vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage,that has the capability of replicating autonomously in a host cell.Vectors typically contain one or a small number of restrictionendonuclease recognition sites at which exogenous DNA sequences can beinserted in a determinable fashion without loss of an essentialbiological function of the vector, as well as a marker gene that issuitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance, ampicillin resistance, orkanamycin resistance.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory regions, including constitutive orinducible promoters, tissue-specific regulatory regions, and enhancers.Such a gene is said to be operably linked to the regulatory regions.

An exogenous gene refers in the present description to a gene that isintroduced into an organism either from a foreign species, or, if fromthe same species is substantially modified from its native form incomposition and/or genomic locus by deliberate human invention. Forexample, any gene, even a structural gene normally found in the hostplant, is considered to be an exogenous gene, if the gene isreintroduced into the organism.

An endogenous gene refers in the present description to a gene that isin its native form and has not been modified in composition or genomiclocus.

A transgenic plant is a plant comprising a DNA region or modification toDNA introduced as a result of the process of transformation.

The term introduced in the context of inserting a nucleic acid into acell, means transfection or transformation or transduction and includesreference to the incorporation of a nucleic acid into a eukaryotic orprokaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (e.g., chromosome, plasmid, plastid or mitochondrialDNA), converted into an autonomous replicon, or transiently expressed(e.g., transfected mRNA).

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A DNA molecule can be designed tocontain a transcriptional template in which the RNA transcript has asequence that is complementary to that of a specific mRNA.

Monocots are a large group of flowering plants, having an embryo withone cotyledon, parts of the flowers usually in threes, leaves withparallel veins and vascular bundles scattered throughout the stem.Examples of monocots include maize, barley, rice, sorghum and wheat.

Dicots are a large group of flowering plants, having an embryo with twocotelydons, parts of the flower usually in twos or fives or multiples,leaves with net veins, and vascular bundles in the stem in a ringsurrounding the central pith. Examples of dicots are tobacco, petunia,canola, sunflower, soybean and tomato.

As used herein, the term plant includes reference to whole plants, plantorgans (e.g., leaves, stems, roots, etc.), seeds and plant cells andprogeny of same. Plant cell, as used therein includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants which can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants. A particularly preferred plant is Zea mays.

T0 refers to the initial transgenic shoot or plant recovered from thetransformation and cultural protocols whether the plant is maintained invitro or established in soil. The T1 generation are those plantsresulting from seed recovered from, most commonly, self pollinated T0plants, or from seed obtained by crosses with other lines where the T0candidate is either the male or female parent. The T2 generation is thematerial obtained from T1 selfings or crosses.

The term oxidase as used in this application refers to an enzyme capableof generating hydrogen peroxide or any reactive oxygen species.

A pathogen refers to any organism responsible for disease and/or damageto a plant. For the present invention, pests include but are not limitedto insects, fungi, bacteria, nematodes, viruses or viroids, parasiticweeds, and the like.

Stress refers to any force that can hurt or damage a plant. Examples ofstress are pathogen attack, invasion by a parasitic weed, environmentalstress such as heat, cold or drought, or mechanical damage. A stressresistant plant is one that is capable of surviving exposure to astress. For example, a sunflower plant expressing oxalate oxidase isable to inhibit the establishment of pathogens, such as Sclerotiniasclerotiorum.

For the purposes of the present invention, a plant that is tolerant to apathogen or other stress is one that is able to withstand a pathogenattack or stressful conditions better than the wild type plant, but willusually succumb to infection and/or die under conditions other than verylight disease or stress pressure. A resistant plant is a plant havingthe ability to exclude or overcome the growth or effects of a pathogenor stress except under extremely high disease or stress pressure. Animmune plant is one capable of complete disease resistance, with noreaction of plant tissue to a potential pathogen.

Plant genera

The hydrogen peroxide/reactive oxygen species producing enzymes incombination with a pathogen tolerant background, as described in thepresent invention can be used over a broad range of plant types,including species from the genera Cucurbita, Rosa, Vitis, Juglans,Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis,Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea,Avena, Hordeum, Secale, Triticum, Sorghum, Picea, Caco, and Populus.

Pathogens

As noted earlier, the hydrogen peroxide/reactive oxygen speciesproducing enzymes of the invention can be utilized to protect plantsfrom insect, disease, and parasitic weed pests. For purposes of thepresent invention, pests include but are not limited to insects,pathogens including ftmgi, bacteria, nematodes, viruses or viroids,parasitic weeds, and the like. Insect pests include insects selectedfrom the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera and Lepidoptera. Insect pests of the invention for the majorcrops include: Maize: Ostrinia nubilalis, European corn borer; Agrotisipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodopterafrugiperda, fall armyworm; Diatraea grandiosella, southwestern cornborer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraeasaccharalis, sugarcane borer; Diabrotica virgifera, western cornrootworm; Diabrotica longicornis barberi, northern corn rootworm;Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotusspp., wireworms; Cyclocephala borealis, northern masked chafer (whitegrub); Cyclocephala immaculata, southern masked chafer (white grub);Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn leafbeetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, cornleaf aphid; Anuraphis maidiradicis, corn root aphid; Blissusleucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus sanguinipes, migratory grasshopper, Hylemya platura, seedcornmaggot; Agromyza parvicornis, corn bloth leafminer; Anaphothripsobscurus, grass thrips; Solenopsis milesta, thief ant; Tetranychusurticae, twospotted spider mite; Sorghum: Chilo partellus, sorghumborer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, cornearworm; Elasmopalpus lignosellus, lesser cornstalk borer; Feltiasubterranea, granulate cutworm; Phyllophaga crinita, white grub;Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cerealleaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorusmaidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Siphaflava, yellow sugarcane aphid; Blissus leucopterus; chinch bug;Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carminespider mite; Tetranychus urticae, twospotted spider mite; Wheat:Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fallarmyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotisorthogonia, pale western cutworm; Elasmopalpus lignosellus, lessercornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata,clover leaf weevil; Diabrotica undecimpunctata howardi, southern cornrootworm; Russian wheat aphid; Schizaphis graminum, greenbug;Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum,redlegged grasshopper; Melanoplus differentialis, differentialgrasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetioladestructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyzaamericana, wheat stem maggot; Hylemya coarctata, wheat bulb fly;Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly;Aceria tulipae, wheat curl mite; Sunflower: Suleima helianthana,sunflower bud moth; Homoeosoma electellum, sunflower moth; Zygogrammaexclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle;Neolasioptera murifeldtiana, sunflower seed midge; Cotton: Heliothisvirescens, cotton boll worm; Helicoverpa zea, cotton bollworm;Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pinkbollworm; Anthonomus grandis, boll weevil; Aphis gossypii, cotton aphid;Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea,bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus differentialis,differential grasshopper; Thrips tabaci, onion thrips; Frankliniellafusca, tobacco thrips; Tetranychus urticae, twospotted spider mite;Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grapecolaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilusoryzae, rice weevil; Nephotettix nigropictus, rice leafhopper; Blissusleucopterus, chinch bug; Acrosternum hilare, green stink bug; Soybean:Pseudoplusia includens, soybean looper; Anticarsia gemmatalis,velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinianubilalis, European corn borer; Agrotis ipsilon, black cutworm;Spodoptera exigua, beet armyworm; Heliothis virescens, cotton boll worm;Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican beanbeetle; Myzus persicae, green stink bug; Melanoplus femurrubrum,redlegged grasshopper; Melanoplus differentialis, differentialgrasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis,soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani,strawberry spider mite; Tetranychus urticae, twospotted spider mite;Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, blackcutworm; Schizaphis graminum, greenbug; Blissus leucopterus leucopterus,chinch bug; Acrosternum hilare, green stink bug; Euschistus servus,brown stink bug; Jylemya platura, seedcorn maggot; Mayetiola destructor,Hessian fly; Petrobia latens, brown seedcorn maggot; Mayetioladestructor, Hessian fly; Petrobia latens, brown seedcorn maggot;Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite;Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid.

Generally Viruses include tobacco or cucumber mosaic virus, ringspotvirus, necrosis virus, maize dwarf mosaic virus, etc. specific viral,fungal and bacterial pathogens for the major crops include: Soybeans:Phytophthora megasperma fsp. Glycinea, Macrophomina phaseolina,Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum,Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii,Cercospora sojina, Peronospora manshurica, Colletotrichum dematium(Colletotrichum truncatum), Corynespora cassiicola, Septoria glycines,Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v.glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythiumdebaryanum, Tomato spotted wilt virus, Heterodera glycines Fusariumsolani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeriamaculans, Rhizoctonia solani, Scierotinia sclerotiorum, Mycosphaerellabrassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum,Alternaria alternata; Alfalfa: Clavibater michiganensis subsp.Insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens,Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma,Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercosporamedicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis,Fusarium oxysporum, Rhizoctonia solani, Uromyces striatus,Colletotrichum trifolii race 1 and race 2, Leptosphaerulina briosiana,Stemphylium botryosum, Stagonospora meliloti, Sclerotinia trifoliorum,Alfalfa Mosaic Virus, Verticillium albo-atrum, Xanthomonas campestrisp.v. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphyliumalfalfae; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystisagropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringaep.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium.graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici,Ascochyta tritici, Cephalosporium gramineum, Colletotrichum graminicola,Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici,Puccinia recondita f.sp. tritici, Puccinia striformis, Pyrenophoratritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae,Pseudocercosporella herptotrichoides, Rhizoctonia solani, Rhizoctoniacerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, BarleyYellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus,Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American WheatStriate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis,Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythiumarrhenomanes, Pythium graminicola, Pythium aphanidermatum, High PlainsVirus, European wheat striate virus; Sunflower: Plasmophora halstedii,Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsishelianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea,Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum,Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Pucciniahelianthi, Verticillium dahlia, Erwinia carotovora pv. carotovora,Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis;Maize: Fusarium moniliforme var. subglutinans, Erwinia stewartii,Fusarium moniliforme, Gibberella zeae (Fusarium graminearum),Stenocarpella maydis (Diplodia maydis), Pythium irregulare, pythiumdebaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum,Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, TCochliobolus heterostrophus), Helminthosporium carbonum I, II & III(Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella zea, Colletotrichum graminicola, Cercospora zeae-maydis,Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora,Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae,Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis,Curvularia pallescens, Clavibacter michiganense subsp. nebraskense,Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak MosaicVirus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomasavenae, Erwinia chrysanthemi pv. Zea, Erwinia carotovora, Corn stuntspiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora sacchari, Spacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium, Maize chlorotic mottle virus, High plains virus, Maizemosaic virus, Maize rayado fino virus, Maize streak virus, Maize stripevirus, Maize rough dwarf virus; Sorghum. Exserohilum turcicum,Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi,Gloeocercospora sorghi, Ascochyta sorghi, Pseudomonas syringae p.v.syringae, Xanthomonas carnpestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Periconiacircinata, Fusarium moniliforme, Alternaria alternate, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara, sacchari, Sporisoriumrelianum (Sphacelotheca reliana), Sphacelotheca cruenta, Sporisoriumsorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Clavicepssorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthonamacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Sclerospora graminicola, Fusarium graminearum, Fusarium Oxysporum,Pythium arrhenomanes, Pythium graminicola, etc.

Generally parasitic weeds include the parasitic flowering plantsOrobanche spp. (Broomrape), the mistletoes (Lorranthaceae: generaArceuthobrium, Viscum, and Phoradendron, dodder (Cuscuta spp.), andStriga spp. (Witchweeds). Parasitic weeds of the present inventioninclude, but are not limited to, Sunflower and Canola: Orobancheaegyptiaca, Orabanche cumana, Tomato and Potato: Orobanche aegyptiaca,Orobanche ramosa, Orobanche cernua, etc.

Hydrogen peroxide/reactive oxygen species producing enzymes

In the present invention, pathogen resistant plants are produced byintroducing into the plant a gene, that codes for an enzyme, whichcauses the production of a reactive oxygen species by an interactionwith an endogenously available substrate. Alternatively, the expressionof an endogenous gene could be altered. Hydrogen peroxide or anyreactive oxygen species may be produced. When hydrogen peroxide isproduced degradation can result in production of reactive oxygen.However, hydrogen peroxide itself may be capable of inducing a stressresponse. Therefore, for purposes of this disclosure, the phrase“reactive oxygen species” is intended to include hydrogen peroxide.There are a number of enzymes that are capable of producing hydrogenperoxide or a reactive oxygen species, for example but not limited to,glucose oxidase, choline oxidase, galactose oxidase, L-aspartateoxidase, xanthine oxidase, monoamine oxidase, eosinophil peroxidase,glycolate oxidase, polyamine oxidase, copper amine oxidase, flavin amineoxidase, berberine Bridge Enzyme, choline oxidase, acyl coA oxidase,amino cyclopropane carboxylate oxidase (ACC oxidase),pyridoxamine-phosphate oxidase, sarcosine oxidase, sulfite oxidase,methyl sterol oxidase, aldehyde oxidase, xanthine oxidase, NADPH oxidase(respiratory burst enzyme homolog), large subunit (GP91) and mostpreferably, oxalate oxidase. It is important in the present inventionthat the transgenic enzyme has available substrate in the plant. In thecontext of exogenous sunflower oxalate oxidase, there is endogenousoxalate present such that, upon expression of the gene, oxalateavailable to the enzyme is subject to degradation resulting in theformation hydrogen peroxide. The expression of genes induced by theunregulated presence of transgene-produced hydrogen peroxide/reactiveoxygen species ultimately results in the accumulation of stressresistance related factors before such stresses are encountered. Thehydrogen peroxide/reactive oxygen species is generated in such a mannerthat the disease or stress response mechanisms of the plant areactivated. If a hydrogen peroxide or reactive oxygen species producingenzyme was selected that did not contain endogenous substrate, the plantcould be transformed with a second gene which upon expression wouldresult in substrate being made. Another option would be to transformplant A with the gene to a hydrogen peroxide or reactive oxygen speciesproducing enzyme in a homozygous state, transform plant B with the geneto the substrate for the enzyme in a homozygous state, and then crossplant A with plant B. The resulting progeny would contain both the geneto the enzyme and the gene to the substrate.

Promoters

In order to express a hydrogen peroxide or reactive oxygen speciesproducing gene, a promoter must be operably linked to that gene. Manydifferent constitutive promoters can be utilized in the instantinvention to express a hydrogen peroxide or reactive oxygen speciesproducing gene. Examples include promoters from plant viruses such asthe 35S promoter from cauliflower mosaic virus (CaMV), as described inOdell, et al., Nature, 313: 810-812 (1985), and hereby incorporated byreference, and promoters from genes such as rice actin (McElroy, et al.,Plant Cell, 163-171 (1990)); ubiquitin (Christensen, et al., Plant Mol.Biol., 12: 619-632 (1992); and Christensen, et al, Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last, et al., Theor. Appl. Genet., 81: 581-588(1991)); MAS (Velten, et al., EMBO J., 3: 2723-2730 (1984)); maize H3histone (Lepetit, et al., Mol. Gen. Genet., 231: 276-285 (1992); andAtanassvoa, et al., Plant Journal, 2(3): 291-300 (1992)), the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the rubisco promoter, the GRP1-8 promoter,ALS promoter, as described in published PCT Application WO 96/30530, asynthetic promoter, such as, Rsyn7, SCP and UCP promoters as describedin U.S. patent application Ser. No. 09/028,819 now U.S. Pat. No.6,042,050 filed Feb. 24, 1998 and herein incorporated by reference, andother transcription initiation regions from various plant genes known tothose of skill.

In the present invention, an expression vector comprises a constitutivepromoter operationally linked to a nucleotide sequence encoding for ahydrogen peroxide/reactive oxygen species producing gene. The expressionvector and an accompanying, selectable marker gene under the directionof a plant-expressible constitutive promoter are introduced into plantcells, selective agent-resistant cells or tissues are recovered,resistant plants are regenerated and T0 candidates are screened forenzyme activity in leaf samples. T0 candidates can also be obtainedwithout the use of a selectable marker. In this instance, the expressionvector is introduced into plant cells without an accompanying selectablemarker gene and transformed tissues are identified and plants screenedbased on enzyme activity alone.

Additional regulatory elements that may be connected to a hydrogenperoxide/reactive oxygen species producing or an oxalate degradingencoding nucleic acid sequence for expression in plant cells includeterminators, polyadenylation sequences, and nucleic acid sequencesencoding signal peptides that permit localization within a plant cell orsecretion of the protein from the cell. Such regulatory elements andmethods for adding or exchanging these elements with the regulatoryelements of the oxalate oxidase gene are known, and include, but are notlimited to, 3′ termination and/or polyadenylation regions such as thoseof the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, etal., Nucl. Acids Res., 12: 369-385 (1983)); the potato proteinaseinhibitor II (PINII) gene (Keil, et al., Nucl. Acids Res., 14: 5641-5650(1986) and hereby incorporated by reference); and An, et al, Plant Cell,1: 115-122 (1989)); and the CaMV 19S gene (Mogen, et al., Plant Cell, 2:1261-1272 (1990)).

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos, et al., J. Biol. Chem., 264:4896-4900 (1989)) and the Nicotiana plumbaginifolia extension gene(DeLoose, et al., Gene, 99: 95-100 (1991)), or signal peptides whichtarget proteins to the vacuole like the sweet potato sporamin gene(Matsuka, et al., Proc. Nat'l Acad. Sci. (USA), 88: 834 (1991)) and thebarley lectin gene (Wilkins, et al., Plant Cell, 2: 301-313 (1990)), orsignals which cause proteins to be secreted such as that of PRIb (Lind,et al., Plant Mol. Biol., 18: 47-53 (1992)), or those which targetproteins to the plastids such as that of rapeseed enoyl-Acp reductase(Verwaert, et al, Plant Mol Biol., 26: 189-202 (1994)) are useful in theinvention. An especially useful signal sequence for this invention issignal sequence isolated from the oxalate oxidase gene. (Lane, et al.,J. Biol. Chem., 266(16): 10461-10469 (1991))

Gene Transformation Methods

Numerous methods for introducing foreign genes into plants are known andcan be used to insert a gene into a plant host, including biological andphysical plant transformation protocols. See, for example, Miki et al,(1993) “Procedure for Introducing Foreign DNA into Plants”, In: Methodsin Plant Molecular Biology and Biotechnology, Glick and Thompson, eds.,CRC Press, Inc., Boca Raton, pages 67-88. The methods chosen vary withthe host plant, and include chemical transfection methods such ascalcium phosphate, microorganism-mediated gene transfer such asAgrobacterium (Horsch, et al., Science, 227: 1229-31 (1985)),electroporation, micro-injection, and biolistic bombardment.

Expression cassettes and vectors and in vitro culture methods for plantcell or tissue transformation and regeneration of plants are known andavailable. See, for example, Gruber, et al., (1993) “Vectors for PlantTransformation” In: Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson, eds. CRC Press, Inc., Boca Raton,pages 89-119.

Agrobacterium-mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants. See, for example,Kado, Crit. Rev. Plant Sci., 10: 1-32 (1991). Descriptions of theAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided in Gruber et al., supra; and Moloney, et al.,Plant Cell Reports, 8: 238-242 (1989).

Direct Gene Transfer

Despite the fact that the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice (Hiei et al., ThePlant Journal, 6: 271-282, (1994)) and maize (Ishida, et al., NatureBiotech., 14: 754-750 (1996)). Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes. (Sanford, etal, Part. Sci. Technol, 5: 27-37 (1987); Sanford, Trends Biotech, 6:299-302 (1988); Sanford, Physiol. Plant, 79: 206-209 (1990); Klein, etal., Biotechnology, 10: 286-291 (1992)).

Another method for physical delivery of DNA to plants is sonication oftarget cells as described in Zang, et al., BioTechnology, 9: 996-996(1991). Alternatively, liposome or spheroplast fusions have been used tointroduce expression vectors into plants. See, for example, Deshayes, etal., EMBO J., 4: 2731-2737 (1985); and Christou, et al., Proc. Nat'l.Acad. Sci. (USA), 84: 3962-3966 (1987). Direct uptake of DNA intoprotoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. See, for example, Hain, etal., Mol. Gen. Genet., 199: 161 (1985); and Draper, et al., Plant CellPhysiol., 23: 451-458 (1982).

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, for example, Donn, et al, (1990) In: Abstracts of theVIIth Int;l. Congress on Plant Cell and Tissue Culture IAPTC, A2-38,page 53; D'Halluin et al., Plant Cell, 4: 1495-1505 (1992); and Spenceret al., Plant MoL Biol., 24: 51-61 (1994).

Particle Wounding/Agrobacterium Delivery

Another useful basic transformation protocol involves a combination ofwounding by particle bombardment, followed by use of Agrobacterium forDNA delivery, as described by Bidney, et al., Plant Mol. Biol., 18:301-31 (1992). Useful plasmids for plant transformation include Bin 19.See Bevan, Nucleic Acids Research, 12: 8711-8721 (1984), and herebyincorporated by reference. This method is preferred for transformationof sunflower plants.

In general, the intact meristem transformation method involves imbibingseed for 24 hours in the dark, removing the cotyledons and root radical,followed by culturing of the meristem explants. Twenty-four hours later,the primary leaves are removed to expose the apical meristem. Theexplants are placed apical dome side up and bombarded, e.g., twice withparticles, followed by co-cultivation with Agrobacterium. To start theco-cultivation for intact meristems, Agrobacterium is placed on themeristem. After about a 3-day co-cultivation period the meristems aretransferred to culture medium with cefotaxime plus kanamycin for theNPTII selection.

The split meristem method involves imbibing seed, breaking of thecotyledons to produce a clean fracture at the plane of the embryonicaxis, excising the root tip and then bisecting the explantslongitudinally between the primordial leaves. The two halves are placedcut surface up on the medium then bombarded twice with particles,followed by co-cultivation with Agrobacterium. For split meristems,after bombardment, the meristems are placed in an Agrobacteriumsuspension for 30 minutes. They are then removed from the suspensiononto solid culture medium for three day co-cultivation. After thisperiod, the meristems are transferred to fresh medium with cefotaximeplus kanamycin for selection.

Transfer by Plant Breeding

Alternatively, once a single transformed plant has been obtained by theforegoing recombinant DNA method, conventional plant breeding methodscan be used to transfer the gene and associated regulatory sequences viacrossing and backcrossing. Such intermediate methods will comprise thefurther steps of: (1) sexually crossing the disease-resistant plant witha plant from the disease susceptible taxon; (2) recovering reproductivematerial from the progeny of the cross; and (3) growingdisease-resistant plants from the reproductive material. Where desirableor necessary, the agronomic characteristics of the susceptible taxon canbe substantially preserved by expanding this method to include thefurther steps of repetitively: (1) backcrossing the disease-resistantprogeny with disease-susceptible plants from the susceptible taxon; and(2) selecting for expression of a hydrogen peroxide producing enzymeactivity (or an associated marker gene) among the progeny of thebackcross, until the desired percentage of the characteristics of thesusceptible taxon are present in the progeny along with the gene orgenes imparting oxalic acid degrading and/or hydrogen peroxide enzymeactivity.

By the term “taxon” herein is meant a unit of botanical classification.It thus includes, genus, species, cultivars, varieties, variants andother minor taxonomic groups which lack a consistent nomenclature.

Sclerotinia Disease

Sclerotinia overwinters as dense, black hyphal masses (sclerotia)deposited in the soil. Sclerotia in the soil germinate when favorableconditions are present to produce mycelial growth for root infections orapothecia for above ground ascospore production. Sclerotinia infectionin sunflower manifests itself in 4 basic forms; basal root mycelialinfection leading to wilt, and middle stalk, bud and head rots. Airborneascospores from soil surface apothecia are responsible for the laterthree infections. The general view has been that Sclerotinia does notinvade healthy tissue but gains a foothold only in wounded areas orsenescing tissue where the spores happen to land. This does not appearto be strictly true, however, in that the only correlation to be madefor successful ascospore infection in plants is the number of hours ofcontinuous moisture to which spores are exposed during the germinationprocess. Anywhere from 24 to 48 hours of damp conditions as well as someminimal level of plant exudate as a nutritional source are required forspore germination and penetration.

Fungal produced oxalate, in conjunction with a host of degradativeenzymes, appears to be a requirement for infection (Noyes, R. D. and J.G. Hancock, Physiol. Plant Pathol., 18(2): 123-132 (1981)). Mutantstrains of Sclerotinia deficient in oxalate production are no longerpathogenic even though other degradative enzymes are produced (Godoy,G., et al., Physiol. Mol Plant. Pathol., 37(3): 179-191 (1990). Inaddition, oxalate fed to sunflower plants exhibit the wilt symptoms ofSclerotinia infection. Therefore, oxalate acts as a classic, diffusabletoxin by stressing host plant tissue in preparation for enzymaticdegradation and mycelial colonization (Maxwell, D. P., Physiol. PlantPathol., 3(2): 279-288 (1973)).

Tolerant backgrounds

The combination of a hydrogen peroxide producing enzyme or an oxalatedegrading enzyme in a plant having a pathogen tolerant geneticbackground yields unexpectedly superior disease resistance compared tothe expression of a hydrogen peroxide producing enzyme or an oxalatedegrading enzyme in a non-tolerant background. Only in combination, doesan immune or near immune plant result.

In the sunflowers expressing oxalate oxidase, oxalate oxidase-producedhydrogen peroxide induces the accumulation in the transgenics of factorsassociated with resistance to stress even though challenges such aspathogen attack are not present. Hydrogen peroxide acts as a signal toinduce the expression of genes involved in stress resistance responsesresulting in plants that stand ready for various challenges prior toencountering such conditions. Unfortunately, one of the side effects ofa continuous, unregulated expression of resistance factors can be alesion mimic phenotype. Lesion mimics are necrotic areas on leaves thatresemble lesions that are present during pathogen assault. Lesion mimicphenotype, however, is not associated with pathogen infection but can berelated to the inappropriate over-expression of genes leading to theaccumulation of stress-related factors such as PR proteins, chitinase,glucanase and salicylic acid. Although not every genotype may produce atransgenic plant with the lesion mimic phenotype, in sunflower, thelesion mimic phenomenon associated with transgenic oxalate oxidaseexpression can be overcome by crossing the transgenic plant with othergermplasm. In the present invention and in the context of Sclerotiniasclerotiorum resistance, oxalate oxidase expressing and lesion mimicexhibiting transgenics when crossed with several different sunflowerinbreds no longer show lesion mimics while still exhibiting oxidaseactivity and varying levels of disease resistance. If such crosses aremade to germplasm that already carries some tolerance to Sclerotinia,then not only is the lesion mimic response eliminated, but the resultingF1 plants are immune to the disease even though the hybrids areheterozygous for the transgene.

In sunflower, favored spore infection sites are the corollas in the headleading to fungal growth into the fleshy receptacle ultimatelydestroying the head, the depression at the nodal attachment point ofpetioles to the main stalk where water can accumulate, leaves and theyoung inflorescence. Root infections and wilt are more common in dryareas of sunflower production and can occur throughout the plant's lifealthough infection from just before flowering to after flowering is mostcommon. Middle stalk and head rots are found in more humid, damp areasand are commonly associated with flowering plants. Thus, tolerantvarieties in sunflower can be tolerant in different parts of the plant.For example, but in no way limited to, the USDA has the followingvarieties of tolerant sunflower germplasm, HA410, HA41 1, HA412, andRHA801.

In canola, Sclerotinia disease is usually a problem only in the stem. Inspring canola, Sclerotinia causes stem rot. In winter canola, stem rotand sometimes root rot can be found. In the canola quality germplasm,currently available, there is no significant stem rot tolerance. Thereis some tolerance in spring canola that originates from Europe(varieties Global, Hanna and Topas), but it is a weak tolerance andcommercially unacceptable. In non-canola quality varieties, a strongerstem tolerance is present in Asiatic germplasm (Newman, et al, Ann. AppLBiol. 110 (Supplement), 8: 150-157 (1987)). However, the stem toleranceis hard to detect, which makes, the current tolerant varieties, usingconventional methods, difficult to develop into canola quality material.Example 2, a partially tolerant canola quality line is crossed with atransgenic line containing the oxalate oxidase gene or the oxalatedecarboxylase gene. For the first time, an enhanced level of tolerancewas seen in the resulting progeny over what can be obtained throughconventional breeding.

In other plants, different levels and types of tolerance can be found.For example, Pioneer has two soybean varieties that are tolerant toSclerotinia. Thus, for the present invention, it would be possible touse different tolerant germplasm as the source to cross with transgenicplants expressing a hydrogen peroxide/reactive oxygen species producingenzyme or an oxalate degrading enzyme or to introduce the enzyme intothe tolerant plant or tissue or to over express an endogenous enzyme.The resulting progeny may have various levels of resistance to thepathogen, because of differing tolerant genetic backgrounds, but asynergistic effect is still seen when a tolerant background is combinedwith the expression of a hydrogen peroxide/reactive oxygen speciesproducing gene or an oxalate degrading enzyme.

In maize, several varieties of maize tolerant to Aspergillus flavus andthe species of Penicillium responsible for ear mold are possiblepathogens that can be used in the present invention. Again, for thepresent invention, it would be possible to use different tolerantgermplasm as the source to cross with transgenic plants expressing ahydrogen peroxide/reactive oxygen species producing enzyme or an oxalatedegrading enzyme or to introduce the enzyme into the tolerant plant ortissue or to over express an endogenous enzyme. The resulting progenymay have various levels of resistance to the pathogen, because ofdiffering tolerant genetic backgrounds, but a synergistic effect isstill seen when a tolerant background is combined with the expression ofa hydrogen peroxide/reactive oxygen species producing gene or an oxalatedegrading enzyme. Examples of Aspergillus tolerant maize varieties arethe Tex6, Y7, Mp420, LB31, L317, C12, N6, 75-R001, B37Ht-2, OH513, andH103 (see Campbell, et al., Plant Disease 79(10): 139-1045 (1995) andCampbell, et al., Phytopathology 85(8):886-896 (1995)).

Introduction of a hydrogen peroxide/reactive oxygen species generatingenzyme or oxalate degrading enzyme into a tolerant background

As described earlier and in the following Example sections, one way ofintroducing a hydrogen peroxide/reactive oxygen species producing enzymeor oxalate degrading enzyme is by transforming a non-tolerant plant withan expression vector containing the enzyme and regenerating plants. Nextthe transgenic plants expressing the enzyme are crossed with a planttolerant to the pathogen.

Alternatively, a tolerant plant or plant tissue could be transformedwith the expression vector containing the enzyme. The resulting plantwould contain both a transgene expressing the enzyme and a geneticallytolerant background.

Another method could be overexpression of an endogenous gene. In someembodiments, isolated nucleic acids that serve as promoter or enhancerelements can be introduced in the appropriate position (generallyupstream) of an endogenous form of the gene(s) encoding an enzyme of thepresent invention so as to up or down regulate expression of thatenzyme. For example, endogenous promoters can be altered in vivo bymutation, deletion, and/or substitution (see: Kmiec, U.S. Pat. No.5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters can beintroduced into a plant cell in the proper orientation and distance froma hydrogen peroxide/reactive oxygen species producing gene so as tocontrol the expression of the gene. Gene expression can be modulatedunder conditions suitable for plant growth so as to alter the enzymecontent and/or composition. Thus, the present invention providescompositions, and methods for making, exogenous promoters and/orenhancers operably linked to a native, endogenous form of an enzyme ofthe present invention.

There are a number of endogenous genes that generated hydrogenperoxide/reactive oxygen species. The following is a list of possibleenzymes whose expression could be altered by the method describedearlier. Oxidoreductases that would be expected to produce reactiveoxygen intermediates such as hydrogen peroxide: Hydrogenperoxide-forming: oxalate oxidase EC 1.2.3.4, glycolate oxidase EC1.1.3.15, polyamine oxidase EC 1.5.3.11, copper amine oxidase EC1.4.3.6, flavin amine oxidase EC 1.4.3.4, berberine Bridge Enzyme EC1.5.3.9, choline oxidase EC 1.1.3.7, acyl coA oxidase EC 1.3.3.6, aminocyclopropane carboxylate oxidase (ACC oxidase), pyridoxamine-phosphateoxidase EC 1.4.3.5, sarcosine oxidase EC 1.5.3.1, sulfite oxidase EC1.8.3.1, and methyl sterol oxidase, Superoxide-forming: aldehyde oxidaseEC 1.2.3.1, xanthine Oxidase EC 1.1.3.22, and NADPH Oxidase (respiratoryburst enzyme homolog), large subunit (GP91).

EXAMPLE 1 SUNFLOWER Cloning of Wheat Oxalate Oxidase

Pioneer wheat variety 2548 (PVP#8900112) was imbibed with running waterfor one hour, wrapped in moist paper towels, sealed in zip-lock plasticbags, and incubated in the dark at 28° C. for 24 hours. Germinating seedwas then frozen in liquid nitrogen and aliquots of 5 to 10 grams werestored at −80° C.

A modified protocol for Castor endosperm RNA isolation was used for RNApurification. (Prescott and Martin, Plant Molec. Bio. Rep., 4(#4):219-224 (1987)) Six grams of frozen germinating seed was ground inliquid nitrogen using a mortar and pestle. Fifty milliliters ofextraction buffer (150 mM LiCl, 50 mM Tris pH 8.0, 5 mM EDTA, 5% (w/v)sodium dodecyl sulfate) was added to the powdered wheat and mixed with50 mls of Phenol/chloroform/isoamyl-alcohol (25:24:1). This was mixed ina Waring blender for one minute. The homogenate was added to 50mIconical centrifuge tubes and centrifuged in a Juoan 412 centrifuge at3000 rpm for 10 minutes. The aqueous phase was then extracted two moretimes with an equal volume of phenol/chloroform/isoamyl-alcohol andfinally with an equal volume of chloroform. The aqueous phase was thenremoved to a baked Corex centrifuge tube and one fourth volume of 10MLiCl added (final concentration approximately 2M LiCl). This was placedat −20° C. overnight. The RNA was collected by centrifugation at 10K for60 minutes and removing all of the supernatant by aspiration withsterile pipettes. The RNA pellet was resuspended in sterile water andquantitated by spectroscopy at OD 260. Gibco/BRL's Superscript FirstStrand Synthesis Kit was used to make first strand cDNA from the totalRNA. The synthesis was primed using oligo dT. All other steps were asstated in the suppliers protocol. PCR was carried out on the firststrand cDNA using oligos

D04244 and D04245.

D04244 is 5′>ggaaggatcctagaaattaaaacccagcggc>3′ (SEQ ID NO: 1)

D04245 is 5′>ccgtcgacaaactctagctgatcaatcc >3′. (SEQ ID NO: 2)

50 μl reaction:

1 μl first strand cDNA

5 μl 10×buffer

1 μl 25 mM dNTPs

1 μl oligo 4244 (1 μg/μl)

1 μl oligo 4245 (1 μg/μl)

1 μl TAQ polymerase

40 μl water

A MJResearch PTC100 thermocycler program was used as follows:

1: 92° C. 1 min.

2: 92° C. 30 sec.

3: 55° C. 30 sec.

4: 72° C. 2 min.

5: Go to step 2 29 times.

6: 72° C. 5 min.

7: 4° C. for ever

8: END

The PCR band that resulted was isolated by gel electrophoresis andpurified by phenol extraction of the DNA from the agarose. This wasdigested with BamHI and SalI for cloning into pGem3ZF+ (Promega,Madison, Wis.) also cut with BamHI and SalI. Ligation of these two DNA'sdid not yield the expected plasmid. A new primer was designed for the 5′end, DO5597, 5′>ccgtcgacaaactgcagctgatcaatcc>3′ (SEQ ID NO: 3).

50 μl reaction:

1 μl first PCR band

5 μl 10×buffer

1 μl 25 mM dNTPs

1 μl oligo 4244 (1 μg/μl)

1 μl oligo 5597 (1 μg/μl)

1 μl TAQ polymerase

40 μl water

A MJResearch PTC100 thermocycler program was used as follows:

1: 92° C. 30 sec.

2: 65° C. 30 sec.

3: Go to 1. 29 times

4: 75° C. 5 min.

5: END

The PCR band that resulted was isolated by gel electrophoresis andpurified by phenol extraction of the DNA from the agarose. This wasdigested with BamHI and PstI for cloning into pGem3ZF+ also cut withBamHI and PstI. Ligation of these two DNA's did yield the expectedplasmid, with one unexpected change. The small polylinker region ofpGem3ZF+ between the BamHI and PstI sites was duplicated on each end ofthe oxalate oxidase cDNA. This resulted in reversing the insert in theparental backbone. This DNA was sent to Iowa State University's NucleicAcid Facility for sequence verification. The only differences are therestriction sites added to the ends by PCR for cloning. The oxalateoxidase protein precursor sequence is illustrated by SEQ ID NO: 4. Theoxalate oxidase cDNA sequence is illustrated in SEQ ID NO: 5.

The plasmids used in this application are pPHP7746 and pPHP8188. PlasmidpPHP7746 contains a pBin19 backbone with two plant transcription unitsbetween TDNA borders. The plant transcription units are 1×CaMV35Spromoter::omega prime leader::oxalate oxidase::pinll terminator and aselectable marker. Plasmid pPHP8188 also contains a pBin19 backbone withtwo plant transcription units between TDNA borders. The planttranscription units are Brassica ALS promoter::oxalate oxidase::pinIIterminator and a selectable marker.

Sunflower Transformation

A general method for transformation of sunflower meristem tissues ispracticed as follows (see also European patent number 486233, hereinincorporated by reference, and Malone-Schoneberg, J., et al., PlantScience, 103: 199-207 (1994)).

Mature sunflower seed (Helianthus annuus L.) of Pioneer(® hybrid 6440 orresearch selection SMF-3 (a selection of USDA germplasm release SFM-3;cms/H. petiolaris Nuttall//cms HA89 backcross) were dehulled using asingle wheat-head thresher. The seed was provided by the Pioneersunflower research station at Woodland, Calif. Seeds were surfacesterilized for 30 minutes in a 20% Chlorox bleach solution with theaddition of two drops of Tween 20 per 50 ml of solution. The seeds wererinsed twice with sterile distilled water.

Split embryonic axis explants were prepared by a modification ofprocedures described by Schrammeijer et al. (Schrammeijer, et al., PlantCell Rep., 9: 55-60 (1990)). Seeds were imbibed in distilled water for60 minutes following the surface sterilization procedure. The cotyledonsof each seed were then broken off producing a clean fracture at theplane of the embryonic axis. Following excision of the root tip, theexplants were bisected longitudinally between the primordial leaves. Thetwo halves were placed, cut surface up, on GBA medium consisting ofMurashige and Skoog mineral elements (Murashige, et al., Physiol Plant,15: 473-497 (1962)), Shepard's vitamin additions (Shepard, (1980) In:Emergent Techniques for the Genetic Improvement of Crops, University ofMinnesota Press), 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5 mg/l6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-acetic acid (IAA), 0.1mg/l gibberellic acid (GA₃), pH 5.6 and 8 g/l Phytagar.

The explants were subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney, et al., Plant Mol. Biol., 18: 301-313(1992)). Thirty to forty explants were placed in a circle at the centerof a 60×20 mm plate for this treatment. Approximately 4.7 mg of 1.8 mmtungsten microprojectiles were re-suspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8) and 1.5 ml aliquots were usedper bombardment. Each plate was bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS 1000® particleacceleration device.

Disarmed Agrobacterium tumefaciens strain EHA105 were used in alltransformation experiments. Binary vectors were introduced into EHA 105using a freeze-thaw transformation method (Holsters, et al., Mol. Gen.Genet., 163: 181-187 (1978)). Bacteria for plant transformationexperiments were grown overnight (28° C. and 100 RPM continuousagitation) in liquid YEP medium (10 μm/l yeast extract, 10 μm/lBactopeptone and 5 μm/l NACl, pH 7.0) with the appropriate antibioticsrequired for bacterial strain and binary plasmid maintenance. Thesuspension was used when it reached an OD₆₀₀ of about 0.4 to 0.8. TheAgrobacterium cells were pelleted and re-suspended at a final OD₆₀₀ of0.5 in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 μm/lNH₄Cl, and 0.3 μm/l MgSO₄.

Freshly bombarded explants were placed in an Agrobacterium suspension,mixed and left undisturbed for 30 minutes. The explants were thentransferred to GBA medium and co-cultivated cut surface down at 26° C.and 18 hour days. After three days of co-cultivation, the explants weretransferred to 374B: (GBA medium lacking growth regulators and a reducedsucrose level of I%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants were cultured for 2 to 5 weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor 1 to 2 weeks of continued development. Explants withdifferentiating, antibiotic resistant areas of growth that had notproduced shoots suitable for excision were transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3 day phytohormonetreatment. Leaf samples from green, kanamycin resistant shoots wereassayed for the presence of NPTII by ELISA and for the presence ofoxalate degrading transgene expression by oxalate oxidase or oxalatedecarboxylase enzyme assays.

NPTII positive shoots were grafted to Pioneer® hybrid 6440 in vitrogrown sunflower seedling rootstock. Surface sterilized seeds weregerminated in 48-0 medium (half strength Murashige and Skoog salts, 0.5%sucrose, 0.3% gelrite, pH 5.6) and grown under conditions described forexplant culture. The upper portion of the seedling was removed, a 1 cmvertical slice was made in the hypocotyl and the transformed shootinserted into the cut. The entire area was wrapped with parafilm tosecure the shoot. Grafted plants could be transferred to soil following1 week of in vitro culture. Grafts in soil were maintained under highhumidity conditions followed by a slow acclimatization to the greenhouseenvironment. Transformed sectors of T₀ plants (parental generation)maturing in the greenhouse were identified by NPTII ELISA and/or byoxalate oxidase or oxalate decarboxylase activity analysis of leafextracts while transgenic seeds harvested from N PTII positive T₀ plantswere identified by oxalate oxidase or oxalate decarboxylase activityanalysis of small portions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedswere dehulled and surface-sterilized for 20 min in a 20 percent Chloroxbleach solution with the addition of two to three drops of Tween 20 per100 ml of solution, then they were rinsed three times with distilledwater. Sterilized seeds were imbibed in the dark at 26° C. for 20 h onfilter paper moistened with water. The cotyledons and root radical wereremoved, and the meristem explants were cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/I GA, and 0.8% Phytagarat pH 5.6) for 24h under the dark. The primary leaves were removed toexpose the apical meristem, around 40 explants were placed with theapical dome facing upward in a 2 cm circle in the center of 374M (GBAmedium with 1.2% Phytagar) and then cultured on the medium for 24 h inthe dark.

Approximately 18.8 mg of 1.8 μm tungsten particles were resuspended in150 μl absolute ethanol. After sonication, 8 μl of it was dropped on thecenter of the surface of macrocarrier. Each plate was bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest was introduced into Agrobacterium tumefaciensstain EHA 105 via freeze thawing as described by Holsters et al., Mol.Gen. Genet. 163: 181-7 (1978). The pellet of overnight grownAgrobacteria at 28° C. in a liquid YEP medium (10 g/l yeast extract, 10g/l Bacto peptone and 5 g/l NACl, pH 7.0) in the presence of 50 μg/lkanamycin was resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH4Cl and 0.3 g/l MgSO4at pH 5.7) to reach an final concentration of 4.0 at OD 600.Particle-bombarded explants were transferred to GBA medium (374E), adroplet of bacteria suspension was placed directly onto the top ofmeristem. The explants were co-cultivated on the medium for 4 days afterwhich the explants were transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets were cultured on the medium for about 2 weeks under 16 h dayand 26° C. incubation conditions.

Explants (around 2 cm long) from two week culture in 374C medium werescreened by oxalate oxidase or oxalate decarboxylase assays. Afteroxalate oxidase or decarboxylase positive explants were identified,those shoots that failed to exhibit oxalate oxidase activity werediscarded, and every positive explant was subdivided into nodalexplants. One nodal explant contained at least one potential node. Thenodal segments were cultured on GBA medium for three to four days topromote the formation of auxiliary buds from each node. Then they weretransferred to 374C medium and allowed to develop for additional fourweeks. Developing buds were separated and cultured for an additionalfour weeks on 374C medium. Pooled leaf samples from each newly recoveredshoot were screened again by the appropriate enzyme assay. At this time,the enzyme positive shoots recovered from a single node will generallyhave been enriched in the transgenic sector detected in the initialassay prior to nodal culture.

Recovered oxidase or decarboxylase positive shoots were grafted toPioneer hybrid 6440 in vitro grown sunflower seedling rootstock. Therootstocks were prepared in the following manner. Seeds were dehulledand surface-sterilized for 20 min in a 20 percent Chlorox bleachsolution with the addition of two to three drop of Tween 20 per 100 mlof solution, and were rinsed three times with distilled water. Thesterilized seeds were germinated on the filter moistened with water forthree days, then they were transferred into 48 medium (half strength MSsalt, 0.5% sucrose, 0.3% gelrite pH 5.0) and grown at 26 ° C. under thedark for 3 days, then incubated at 16 h day culture condition. The upperportion of selected seedling was removed, a vertical slice was made ineach hypocotyl, and a transformed shoot was inserted into a V-cut. Thecut area was wrapped with parafilm. After one week culture on themedium, grafted plants were transferred to soil. In the first two weeks,they were maintained under high humidity conditions to acclimatize agreenhouse environment.

Transformed sectors of T0 plants were identified by additional oxalateoxidase or decarboxylase assays of those in vitro positive graftedshoots. After assay, non-transformed sectors were trimmed off andauxiliary buds from transgenic sectors were recovered so as to obtainnear uniform transformation events. Selfed seed from T0's werecollected, germinated, characterized for enzyme activity, and selfedagain.

Oxalate Oxidase Expression in Sunflower Transgenics

A small set of wheat oxalate oxidase expressing sunflower transgenicswere forwarded to the Pioneer sunflower breeding station at Woodland,Calif. for Sclerotinia disease resistance evaluation. A mycelialinoculation protocol was developed where the pathogen was introduced tothe plant through small pieces of Sclerotinia infested carrot. A freshlyinfected carrot slice was placed on a petiole midway between the stemand leaf. A parafilm wrap was applied to hold the carrot piece in placeand to maintain a high humidity environment at this junction. Threemiddle level petioles/plant were inoculated on greenhouse grown plantsprior to first ring anthesis. The carrot piece was removed 24 hourslater and disease progression was monitored as the fungus moved into thestem. Approximately 3 weeks later a visual rating was taken of thelesions on the stem and given a value of 1 to 9 where 1 representedtypical susceptible lesions and 9 denoted a high degree of resistance.The same readings were done 7-8 days later. In addition, the inoculatedplants were collected at dry down, the main stems split and the fungalsclerotia bodies were recovered, weighed and counted.

The initial set of 5 oxalate oxidase expressing T2 transgenics (lineSMF-3) recovered from pPHP7746 transformations showed remarkable diseaseresistance responses following fungal challenge. While adequatequantitative data to correlate oxidase activity with resistance ratingswere not collected with this first set of plants, it was visuallyapparent that the transgenics were significantly more resistant toSclerotinia mycelial infection than the nontransgenic controls(nontransformed SMF-3). Disease would easily progress down the petioleof all inoculated plants, however, in a number of the transgenicsfurther progression into the stem was either restricted or halted at thestem. There were indications that the higher expressing oxidasetransgenics inhibited disease progression to a greater extent than lowerexpressing individuals. In the end, all nontransgenic plants were deadwhile many of the transgenics were able to survive to produce seed.Although the plants showed significant disease resistance, the plantsthemselves had numerous disease-like lesions on their leaves. Thisphenomena is often seen in mutants that have a non-functional ormodified gene in the disease resistance pathway. Although the overallhealth and seed yield of the sunflower plants did not seem to bestrongly affected under greenhouse conditions, plant with lesions wouldnot be commercially viable and may not maintain their yield in fieldconditions.

Infection experiments with this initial set of transgenics have beendesigned to determine the correlation between transgene activity anddisease resistance response. The results are with a set of T2 plants ofthe best performing event in the initial trial, #193870. Data from 19transgenic plants and 23 controls were collected following fungalinoculation. On the day of inoculation, the leaf and part of the petioleabove the inoculation site of each treated petiole were removed, andshipped to Johnston, overnight express, on wet ice. The samples werelyophilized to dryness and equal portions of petiole or leaf associatedwith each of the 3 inoculated petioles were pooled and ground to a finepowder. Oxidase enzyme assays (Suigura, et al., Chem. Pharm. Bull.,27(9): 2003-2007 (1979) and hereby incorporated by reference) wereperformed on each leaf and petiole sample. The oxidase enzyme assays areas follows: (1) Leaf tissue was lyophilized and grind to a fine powder.The powder was resuspended in Na-succinate buffer (0.1M, pH 3.5)+a dropof Tween-20 at a 1 mg/ml concentration; (2) Individual 1 ml reactionswere set up in tubes or a larger volume reaction mix in a small beakerwith stirring for a time course. Into each tube: Tube assay-100 μl ofsuspension, 100 μl of 10 mM oxalate in 0.1M Na-succinate buffer, pH 3.5,succinate buffer to bring volume to 1 ml. Tissue extract was added lastand this started the reaction timing. The reaction was allowed toproceed for a defined time (1-30 minutes) with agitation and 100 μl ofreaction mix was removed to microtitre plate wells that contain 17.5 μlof 1M Tris free base. Then 82.5 μl was added of the peroxidase-linkedcolor development solution (8 mg 4-aminoantipyrine, 20 μlN,N-dimethylaniline, 400 μl of peroxidase all in 100 ml of 0.2MTris-HCl, pH 7.0). The absorbance was read at 550 nm. For the timecourse assay, successive 100 μl aliquots were removed from the 1 mlreaction tube at the desired times. Time vs. absorbance was plotted anda slope was determined (OD550/min.). This value based on the initial dryweights can be used to compare different samples and plants.

The enzyme assay results are presented as either a slope of a timecourse reaction, A550/minute, or as a “specific activity” calculatedfrom a slope (mM oxalate converted/minute/mg powder). A summary table isas follows:

TABLE 1 Avg. oxidase activity (mM (1 = sus., Scler- oxalate/ 10 = res.)otia Scler- min/mg) Lesion- Lesion- wt. otia # petiole leaf 1 2 (gm) #Controls 23 0   0 2.2 1.1 4.0  84   193870 19 71.6 119 7.6 6.1 0.43 11.4

The transgenics exhibited not only a significant improvement in thesubjective disease ratings compared to the nontransgenic controls, butalso showed a 10 fold decrease in the amount of fungal formed sclerotiadeposited in the stems. 35S oxalate oxidase expression effectivelydisrupts a middle stalk rot-type of mycelial Sclerotinia invasion bydramatically reducing the rate of movement of the disease front throughtissue as well as impacting the ability of the fungus to produce storagebodies that would serve as inoculum in subsequent crop cycles.

In addition to the observations with the 193870 T2 transgenic fourtrials of a small set of 35S::oxalate oxidase transgenics transformedwith pPHP7746 were performed.

The preferred measure of disease resistance in sunflower is sclerotiaaccumulation. The complex interaction of host and pathogen throughoutthe life cycle of both is distilled down to one measurement. The abilityof the fungus to propagate itself will depend on health of the pathogenand the extent of the fungal invasion of the host. Poor diseaseestablishment is reflected in the inability of the fungus to“reproduce.” FIG. 1 shows that oxalate oxidase expression significantlyreduces the number of sclerotia in oxalate oxidase expressing plants.FIG. 2 shows that oxalate oxidase expression significantly reduced themass of sclerotia produced in oxalate oxidase expressing plants.

FIG. 3 is the plot of the % of oxalate oxidase expressors or negativecontrols vs. ranges of sclerotia harvested at the end of the testingcycle and reveals the significant impact oxidase expression has ondisease resistance. Clearly, FIG. 3 shows that sclerotia weightssignificantly decrease in oxalate oxidase expressors. In fact, themajority of oxalate oxidase expressors contained less than 0.5 gm ofscelortia. FIG. 4 demonstrates the correlation of the level of oxidaseactivity has on sclerotia weight and thus on disease resistance. Ingeneral, the higher the oxidase expression, the less sclerotia areformed. Thus, oxidase expression prevents the formation of sclerotia.

A significant observation is that an important consequence of oxalateoxidase expression in sunflower is high levels of pathogenesis-relatedfactors (PR) accumulate in the plants in the absence of pathogenchallenge. Examples of PR factors include PR-1, chitinase, 14-3-3protein, and glucanase. Leaf tissue was pulverized in 24 mM sodiumphosphate-citrate buffer, pH 2.8 containing 6 mM L-ascorbic acid and 14mM 2-mercaptoethanol. The homogenate was centrifuged and solubleproteins in the supernatant were analyzed by denaturing polyacrylamideelectrophoresis followed by Western blotting according to Towbin, etal., Proc. Nat'l. Acad. Sci. (USA), 76: 4350 (1979) and Anderson, etal., Electrophoresis, 3: 135 (1982). Blots were probed with rabbitantisera raised against purified tobacco PR1b (kindly provided by Dr.Ray White, Rothamsted Experimental Station, Harpenden, Herts, UK),glucanase, 14-3-3 protein, or chitinase. Anti-glucanase andanti-chitinase sera were obtained from rabbits inoculated with E. coliexpressed GST-glucanase or GST-chitinase fusion proteins. Arabidopsisanti-14-3-3 antibodies were uses to determine the presence of 14-3-3protein in sunflower extracts. After incubation with primary antiserum,protein blots were treated with alkaline phosphatase-conjugatedsecondary antibodies, washed, and analyzed by chemiluminescence(Western-Light, Tropix). Western analysis for tobacco PR-1, chitinase,14-3-3, and glucanase of unchallenged sunflower oxidase transgenic193870 leaf extracts revealed significantly increased levels of all fourrelative to the nontransgenic control.

Salicylic acid levels in the oxidase-expressing line were also studied.Free and total (free plus conjugated) SA was extracted from 0.4 g leafsamples as previously described (Enyedi, et al., Proc. Natl. Acad Sci.(USA), 89: 2480-2482 (1992)). Samples were analyzed with a Waters liquidchromatography system (Waters Corp., Milford, Mass.). Ten microliters ofeach extract were injected at a flow rate of 1 ml/min into a Nova-Pak 4μm C-18 column (3.9 cm×75 mm; Waters Corp.). The column was maintainedat 40° C. and equilibrated in 22% acetonitrile against 78% of 0.1%acetic acid in water. SA was eluted isocratically under these conditions(Rt 3.1 min) and quantified using a scanning fluorescence detector(model 474, Waters Corp.) using excitation and emission wavelengths of300 and 405 nm, respectively. The identity of SA in sunflower extractswas confirmed by its co-elution with authentic standard and by analysisof its UV light absorption spectrum, as measured with a photodiode arraydetector (model 996, Waters Corp.). SA levels in the oxidase-expressingline were 6 fold that of the control.

A larger set of leaf samples from the prior greenhouse evaluations wereexamined for PR-1 levels and expression and quantified by assigning a 0thorough 3+ for the banding intensity. This information was plotted vs.sclerotia accumulation to determine if increased levels of PR proteinwould be useful as a disease resistance predictor. (FIG. 5) It seemsclear that PR-1 is present during disease resistance and that oxalateoxidase expression is able to induce this factor through the generationof hydrogen peroxide by the degradation of cellular, not fungal,substrate. Other observations are that the PR-1 rating of 1+ is notsignificant from 0, 75% of the oxidase transgenics have PR-I ratings of2+ or 3+, and 70% of the non-oxidase controls have PR-1 ratings of 0 or1+. In addition when oxidase activity is plotted against the PR-1ratings (FIG. 7). a direct correlation between oxalate oxidase levelsand PR-1 protein amounts can be seen. Increased expression of oxidaseoxal ate causes the level of PR-1 protein to also increase.

To address the relationship between oxalate oxidase activity, SA levelsand PR protein levels, a time course study was carried out with twotransgenic lines of sunflower expressing oxalate oxidase(193870-1X35S::oxalate oxidase and 610255-SCP1::oxalate oxidase). Theresults consistently showed that oxalate oxidase activity rapidlyincreased to a high level (greater than 300 μM/Min.mg) within 4 weeksfrom germination in transgenic plants. No activity was detected incontrol SMF3 seedlings. From 2 to 4 weeks SA and PR-1 levels are similarto control plants. Yet by 6 weeks in transgenic plants a 2-4 foldincrease in the level of SA and PR1 can be seen as compared to controls.At 6 weeks from germination both SA and PR-1 were significantly inducedin oxalate oxidase expressing plants in the absence of pathogenchallenge.

The function of PR-1 protein is largely unknown. The determine thepossible effect of PR1 protein on Sclerotinia, an in vitro assay usingpurified tobacco PR1 was carried out. A suspension of 300 Sclerotiniaspores in 100 μl of a solution of 13% sucrose, with or without PR-1, wasplaced in a micro-plate. The spores were incubated at room temperaturefor 24 hours. Following the 24 hour incubation the micro-plates wereexamined microscopically. Germination was defined as the appearance ofScwerotinia hyphae in the suspension. At 1.6 μM PR-1b minimal inhibitionwas observed. At 4.9 μM PR-1b no hyphal growth was found and completeinhibition was seen. Thus, induction of PR-1 in a plant will restrictScierotinia spore germination.

Oxalate Decarboxylase Expression in Sunflower Transgenics

The oxalate decarboxylase gene isolated from Flammulina (WO 94.12622published in Jun. 9, 1994 and hereby incorporated by reference) wasintroduced into sunflower, as described earlier, and Scperotinia diseasescreenings were performed using the same protocols of inoculation andrating employed for oxidase transgenics. Optionally, another gene foroxalate decarboxylase can be used. The oxalate decarboxylase geneisolated from Aspergillus phoenices (U.S. patent application Ser. No.08/821,827, filed on Mar. 21, 1997) may also be used in the presentinvention. Expression of oxalate decarboxylase in transformed plantcells and tissues can be detected by the enzyme assay described byLabrou, et al, J. Biotech, 40: 59-70(1995); and Johnson, et al.,Biochem. Biophys. Acta 89:351 (1964). Decarboxylase activity is linkedto a second activity, that of formate dehydrogenase, that will oxidizethe decarboxylase generated formate with the subsequent reduction of NADto NADH (Johnson, et al., Biochem Biophys. Acta 89:351 (1964)). Theincrease of OD340 as NAD is reduced is used to generate an initialreaction rate that is linear with respect to formate concentration from0.2 to 2.0 umole. The assay was adapted to megatiter plates so that alarge number of samples could be screened. Leaf tissue samples wereprepared and assays set up as in the oxalate oxidase assay protocolexcept that the decarboxylase reactions were run from 1 to 3 hours.Following the assay period, 100 μl of each reaction supernatant weretransferred to microtitre plate wells and 17.5 μl of 1M Tris free basesolution was added to each. Next 10 μl of b-NAD (6.6 mg/ml stock, Sigma)are added to each sample well and mixed followed by the addition of 5 μlof formate dehydrogenase (4.0 mg/ml stock, 1 enzyme unit/mg solid,Sigma). The absorbance at 340 nm was measured repeatedly over a 10minute period to generate a reaction rate curve. The slope of theinitial rate curve was determined.

Replicated greenhouse disease screens of several Flammulinadecarboxylase-expressing transgenics demonstrated that decarboxylasemetabolism of oxalate is an effective deterrent to sclerotinia diseaseprogression in sunflower, as can be seen in Table 2.

TABLE 2 Line Gene Sclerotia wt. Avg. (gm) 436741 decarboxylase 0.43436747 decarboxylase 0.65 436784 decarboxylase 1.14 436705 decarboxylase0.13 436724 decarboxylase 0.43 460078 decarboxylase 0.95 193870 oxidase0 SMF-3 control 0.73

The correlation of decarboxylase activity with sclerotia accumulation ispresented in FIG. 7. In general, the higher the expression ofdecarboxylase the less sclerotia are formed. In order to understand therole of oxalate degradation and the generation of the disease response,oxalate decarboxylase transgenic material was tested for the presence ofPR-1 proteins as previously described. As can be seen in FIG. 8 there islittle correlation between oxaxlate decarboxylase activity and thepresence of PR-1. Thus, degradation of oxalate by oxalate decarboxylasedoes prevent Sclerotinia disease progress, but does not initiate thedisease response pathway as seen in oxalate oxidase transgenic plants.

Combining Conventionally Tolerant Sunflower Lines with Oxalate Oxidaseor Oxalate Decarboxylase Expressors

In order to cross the oxalate oxidase or oxalate decarboxylase gene intoconventionally tolerant sunflower lines, the following procedure wasperformed. Pollen was collected from the best challenged event, 193870.A single head from 8 different elite lines were selected and handemasculated daily. This entails removing the anthers from the ring offlowers that emerge each day and rinsing with water to ensure no pollenremains that could self-pollinate. This procedure was done for 3-4 daysupon which time the center of the head is cut out, leaving no moreflowers to emasculate. The pollen, from the donor plant was collected ona paper towel, crossing paper and then rubbed on the emasculated heads.All flowering heads were bagged before and after any crossing. Theresulting progeny were grown in the greenhouse and tested for resistanceto Sclerotinia and evidence of the lesion mimic phenotype.

Oxalate oxidase-produced hydrogen peroxide induces the accumulation offactors associated with resistance to stress even though challenges suchas pathogen attack are not present. Unfortunately, one of the sideeffects of a continuous, unregulated expression of resistance factors isa lesion mimic phenotype. Sunflower oxalate oxidase expressing plantsshow numerous necrotic lesions on their leaves. In FIG. 9 the oxidaseactivity in transgenic plants is compared to the lesion rating. The datapoints are the averages over 4 replications performed throughout theyear. The ratings are as follows: 1=susceptible, up to 9=resistant. Innon-transgenic plants lesions are a sign of a disease infection. In thecase of oxalate oxidase expressing transgenics, that were highlyresistant to disease, there were still a large number of lesions.

By crossing oxalate oxidase expressing plants with germplasm showingtolerance to Sclerotinia a sunflower plant with immunity to Sclerotiniaand no lesion mimic phenotypes were produced. The combination of oxalateoxidase expression in a tolerant germplasm background causes asynergistic effect of a superior plant which is far better than eitherof its parents. Table 3 summarizes the data for this set of experiments.PR126M and PR 118M are both sunflower lines which show tolerance toSclerotinia. PK93M and PK68F are susceptible to Sclerotinia infections.

TABLE 3 oxox Lesion Sclerotia Line Activity Rating wt. (gm) # transgenic193870 0.082 7.1 0.8 27 PR126M (T) × 193870 0.073 8.0 0    0 PR118M (T)× 193870 0.065 8.3 0    0 PK93M (S) × 193870 0.066 6.3 1.0 48 PK68F (S)× 193870 0.049 3.5 2.0 66

As can be seen in Table 3, all the plants showed comparable levels ofoxalate oxidase expression, yet there were no Sclerotia formed in theoxalate oxidase expressors crossed with tolerant germplasm. In addition,the lesion levels were the least for the oxalate oxidase expressorscrossed with tolerant germplasm. In the absence of Sclerotinia infectionthe oxalate oxidase expressors crossed with the tolerant germplasmplants exhibited a normal leaf phenotype with no necrotic lesions. Thus,the synergistic effect of oxalate expression and tolerance geneticsproduces a commercial quality plant, immune to Sclerotinia infection butwith no negative phenotypes.

In order to demonstrate that the greater resistance of the transgenic,Sclerotinia tolerant sunflower is the result of the presence of theoxalate oxidase gene, the four lines listed in Table 3 were used ingenetic crosses designed to create equivalent materials which contain orlack the transgene. The Sclerotinia inoculations were done using thesame technique as that described to generate the data in Table 3. Thetest was allowed to proceed to the end of the sunflower life cycle atwhich time the dried stalks were obtained and individually split open inorder to isolate Sclerotinia sclerotial bodies. A more invasive orextensive mycelial mass in an infected plant is an indication of a moresuccessful infection and leads to increased sclerotial body production.The sclerotial body number and total weight were used to measure thebiological success and reproductive fitness of Sclerotinia and,therefore, of the ability of sunflower lines to inhibit diseaseprogression and decrease fitness.

FIG. 10 presents the sclerotial body weights from all of the linestested. Each bar on the bar graph represents the mean and standard errorof eight individual plants. The individual plants are described in thefollowing Table 4. The numbers labeling each bar of FIG. 10 correspondto the specific genotype and whether or not it is transgenic accordingto the Table 4.

TABLE 4 Number Line  1. Susceptible Check 1  2. SMF3 (sus*)  3. SMF3(sus)  4. USDA 894 (sus)  5. PK68F (sus) X SMF3  6. PK93M (sus)  7.PR118M (tol#) X SMF3  8. Susceptible Check 2  9. PK93M (sus) X SMF3 10.PR126M (tol) X SMF3 11. Event 193870 (SMF3 transgenic) 12. PK68G (sus)13. Event 610255 (SMF3 transgenic) 14. PR118M (tol) 15. PK68F (sus) X193870 16. PR118M (tol) X 193870 17. PR126M (tol) 18. PK93M (sus) X193870 19. Event 539149 (SMF3 transgenic) 20. PR126M (tol) X 193870*sus—susceptible to Sclerotinia infection. #tol—tolerant to Sclerotiniainfection.

Data presented in this graph directly support conclusions made from datapresented in Table 3. Most importantly, the genetic Sclerotiniatolerance combined with the oxalate oxidase transgene gave a near immuneresponse to this pathogen (see FIG. 10, bars 16 and 20) which issuperior to the resistance of either parent. The increased resistancewas not due to higher oxalate oxidase enzyme activity since thesusceptible and tolerant line crosses with 193870 gave comparableactivity levels (Table 5). In fact, 193870 is a homozygous line anddemonstrated about twice the activity of the heterozygous crosses.Susceptible check lines showed the highest sclerotial body weightsbecause the fungus growth was relatively uninhibited (FIG. 10). Tolerantlines, transgenic lines, and susceptible lines crossed with event 193870were superior to non-transgenic susceptible lines, and susceptible ortolerant lines crossed with non-transgenic SMF3.

TABLE 5 Oxalate oxidase enzyme activity in transgenic SMF3 plants and inhybrid plants resulting from genetic crosses between SMF3 andSclerotinia susceptible or tolerant parents. Oxalate oxidase activityLine Genetic Status Parent Line (OD550/mg/hr) Event 193870 homozygousSusceptible 53 Event 610255 homozygous Susceptible 66 PK68F X 193870heterozygous Susceptible 22 PK93M X 193870 heterozygous Susceptible 29PR118M X 193870 heterozygous Tolerant 30 PR126M X 193870 heterozygousTolerant 30

When non-transgenic hybrids are compared to parental lines somedifferences in the line contribution to resistance can be observed. Bothtolerant line hybrids showed greater sclerotial body weights than theparental tolerant line alone, suggesting that the cross with SMF3 mayhave reduced their ability to resist Sclerotinia (FIGS. 11-14). ThePK93M hybrid had a similar to slightly reduced sclerotial weight valuecompared to parental line PK93M. In this case, there may be a slightcontribution to resistance made by SMF3 or perhaps hybrid vigor. Thesame case might be made for the non-transgenic hybrid with parental linePK68F. This hybrid has significantly lower values than the SMF3 parent.The. PK68F parental line, however, was not included as a control in thatdata set so no direct comparison could be made. Parental line SMF3 hadhigher sclerotial body weights and numbers than any of the other linesto which it was crossed. These comparisons suggest that there may be aslight SMF3 contribution to Sclerotinia resistance in the susceptibleline hybrids but that it may actually reduce resistance in the tolerantlines.

Comparisons made between transgenic SMF3 event 193870 (SMF3/35Soxox inFIGS. 11-14) and the parental non-transgenic lines show that it is thesuperior line for resistance compared to susceptible line PK93M, butthat the Sclerotinia tolerant, non-transgenic parents are moreresistant. When compared to non-transgenic SMF3 it is clear that theoxalate oxidase transgene contributes significantly to resistance. Theremay be conventional genetics which are more resistant to Sclerotiniathan oxalate oxidase transgenic SMF3, but FIGS. 11-14 demonstrate thatthe presence of this transgene enhances resistance in every line inwhich it was introduced. When combined with the tolerant genetics ofPR126M, oxalate oxidase conferred nearly complete immunity toSclerotinia (FIGS. 11-14). Six out of the eight PR126M/193870 plants hadno sclerotial bodies at all (data not shown). The resistance of thisline surpassed all of the others presented in spite of the fact that ithas lower enzyme activity than 193870 and that SMF3 may detract from thegenetic resistance. This data confirms the synergistic effect of theoxalate oxidase gene and Sclerotinia tolerant genetics.

Transgenic oxalate decarboxylase expressing plants are crossed asdescribed above, into conventionally tolerant backgrounds. Oxalatedecarboxylase expressing plants are tested for resistance to Sclerotiniaas described earlier.

EXAMPLE 2 CANOLA

Sclerotinia stem rot is a disease of Brassica napus, Brassica rapa andBrassica juncea caused by the fungus Sclerotinia sclerotiorum.Sclerotinia stem rot is also found in more than 400 other dicots. Thedisease results in premature ripening and seed shriveling in the field.Canola is a Brassica rapeseed crop with low glucosinolates and lowerucic acid content. All cultivars/hybrids of canola quality grownworldwide are susceptible to this disease. Brun, H., et al., pp.1216-1221. Proceedings of the 7^(th) International Congress on Rapeseed,Poznan, (1987).

Partial conventional tolerance was found in Asiatic germplasm. Currentattempts to transfer this tolerance into canola quality material andattain enhanced tolerance have been unsuccessful, primarily due to thecomplexity of the disease, a low tolerance level and a hardship inscreening to detect tolerance in breeding prograrns. Another approachtaken was to transform canola plants with the oxalate oxidase gene whichdegrades oxalic acid generated by the fungus into hydrogen peroxide andcarbon dioxide. For the first time an enhanced level of tolerance to thefungus was seen in the F1's generated from crosses of a conventionallytolerant canola quality line and a transgenic line containing theoxalate oxidase gene.

Canola Transformation Protocol

The following is a standard procedure of Agrobacterium (strain LBA4404)mediated cotyledonary transformation (Moloney, M. et al, Plant CellRep., 8: 238-242 (1989)) used to produce 16 pPHP7746 and 64 pPHP8188transgenics B. napus Pioneer variety 46A65.

1. Sowing seed for experiments

1. Place approximately 250 seeds in seed sterilization apparatus.

2. Spray seeds with 70% ETOH.

3. Place seed apparatus in 2% sodium hypochlorite solution (30%commercial bleach solution) and let sit for 15 minutes, stirringoccasionally to ensure seeds are thoroughly soaked.

4. Place seed apparatus in sterile water for 5 minutes to rinse offbleach from the seeds.

5. Empty out seeds into a sterile petri dish.

6. Plate seeds onto GM (germination medium) (Murashige and Skoog salts(MS salts, Gibco) 10 mls/liter of MS supplements for organics (in 1liter, 10 g of I-inositol, 40 mg Thiamine-Hcl and 100 g MES), 3%sucrose, pH 5.8, and 0.2% gelrite), at 10-12 seeds/plate. Box plates.Place box in 10° C. to synchronize germination or may place directly in24° C. (tissue culture room).

7. Seedlings ready for cocultivation in 4-6 days (dependent oncultivar).

2. Grow Agrobacterium containing gene of interest

1. Obtain Agrobacterium with gene of interest from vector constructionprovider. Used pPHP7746 and pPHP8188.

2. Prepare bacterial overnight culture 1 day prior to cocultivation, byculturing 10 μl loop of Agrobacterium in 15-30 ml of LB broth (GIBCO)supplemented with appropriate antibiotics and 200 uM acetosyringone.

3. Place culture tube/flask in 28 C shaker at 200 rpm.

4. On day of cocultivation, centrifuge the bacterial suspension at2000-3000 rpm for 10 minutes.

5. Discard supernatant and then resuspend bacterial pellet in 15-40 mlof MS-H medium (MS salts (Gibco) 10 ml/liter MS supplements for organics(see above), 2% sucrose, and pH 5.7) containing 200 uM acetosyringone.

6. Decant suspension into 30 ml sterile petri dishes, between 5-15ml/dish.

3. Napus: Cocultivation

1. Excise cotyledons such that as much of the petiole is intact aspossible without including the meristems.

2. Plate 1 non-inoculated control plate, using 10-12 cotyledons, withthe remaining cotyledons, dip the cotyledonary petiole (cut end) intothe bacterial suspension (2.6)

3. Plate the inoculated cotyledons on MMW media (MS salts (Gibco), 10ml/liter MS supplement for organics (see above), 3% sucrose, 4.5 mgbenzladeninepurine (1st dissolved is the least possible amount ofmethanol), 0.1 mg/liter abscissic acid, pH 5.8, and 6% agar (Sigma#1296) at 20-30 cotyledons/plate.

4. Wrap all plates with surgical tape, and place in transparent box intissue culture room at 24 C, for 2-3 days with 16 hour photoperiod.

Transfer cotyledons as follows:

1. Non -inoculated control—5-6 cotyledons on MMW media (MS salts(Gibco), 10 ml/liter MS supplement for organics (see above), 3% sucrose,4.5 mg benzladeninepurine (First dissolved is the least possible amountof methanol), 0.1 mg/liter abscissic acid, pH 5.8, and 6% agar (Sigma#1296)+carbenicillin 300-500mg/L, and on MMW+carbenicillin 300-500mg/L+selection agent. (Kan 100 mg/L for pPHP7746 and Glufosinate 4mg/Lfor pPHP8188)

2. Inoculated control—5-6 cotyledons on MMW+carbenicillin (carb) 300-500mg/L

3. All remaining cotyledons—plated on MMW+carb 300-500 mg/L+selectionagent (see above)

4. All plates are sealed with surgical tape, boxed, and place in tissueculture room for 3 weeks.

Second transfer to shoot selection media:

1. Transfer cotyledons onto media as above. Seal plates, box and culturefor an additional 3 weeks, or until shoots form from cut end.

2. Excise green, healthy shoots and place on B5-H H (see media recipebelow)+carb 300-500 mg/L+selection agent media. This media allowsrooting.

3. If shoots have rooted, assay for reporter gene activity.

4. After analysis, transplant confirmed transgenic shoots to soil.

B5-H Media Ingredients 5 LITERS Combine: B5 x5 stock 1 L Sucrose (2%)100 g USE filtered H₂O to up volume to 5 L Phytagel (Sigma #p8169) 20 gpH solution to 5.8 Autoclave After Autoclaving: Add appropriateselective agents or plant hormones B5 Bx Stock Stock Ingredients 4LITERS Combine: Potassium Nitrate (KNO₃) 50.0 g Magnesium Sulphate(MgSO₄—7H₂O) 5.00 g Calcium Chloride Dihydrate (CaCl₂2H₂O) 15.00 gAmmonium Sulphate ((NH4)₂SO₄) 2.68 g Sodium Phosphate Monobasic(NaH₂PO4-H₂O) 3.00 g Iron 330 (Fe330) 0.80 g B5 Vitamin Stock (100x) 200mls B5 Micronutrients (100x) 200 mls Potassium Iodide Stock 20 mls Bringup the volume to 4 L with filtered water B5 Vitamin Stock (100x)Ingredient 1 LITER Combine: Myo-inositol 10.0 g Nicotinic Acid 100.0 mgPyridoxine HCL 100.0 mg Thiamine HCL 1.0 g Bring up the volume to 1 Lwith filtered water Micronutrient Stock Solution (1000x) Ingredient 1LITER Combine: Manganous Sulphate (MnSO₄—H₂O) 10.0 g Boric Acid (H₃BO₃)3.0 g Zinc Sulphate-7 Hydrate (ZnSO₄—7H₂O) 2.0 g Sodium Molybdate(Na₂MoO₄—2H₂O) 250.0 mg Cupric Sulphate-5 Hydrate (CuSO₄—5H₂O) 25.0 mgCobalt Chloride-6 Hydrate (CoCl₂—6H₂O) 25.0 mg Bring up the volume to 1L with filtered water

Make to 100× before using by:

Adding 100 ml of 1000× and bringing it up to a 1 Litre volume by adding900 ml of filtered water

Potassium Iodide (KI) Solution

Add 0.83 g of KI in 1 litre of filtered water.

Two of the sixteen transgenics were single copy integration events.Progeny (T1) from these single events along with three double copy linesand two multiple copy lines underwent greenhouse Sclerotinia screening.Results from the indoor screening showed the double copy line 170B3 andsingle copy line 164B1 were more disease tolerant than theirnon-transformed parent line NS1565. Oxidase assays results on the T2transgenic plants screened showed 170B3 and 164B1 plants had the highestand second highest expression levels respectively when compared againstthe other T1 lines tested. The 164B1 and 170B3 T2 lines were fieldtested.

Field Screening of Oxalate Oxidase Canola Transgenics

Field trials were conducted at the Stirk's site in Hillsburg, Ontario,Canada. The level of background inoculum was high and it was assessed byvisual estimate of the presence of apothecia and by doing a petal test.A petal test is performed by placing canola petals on Potato DextroseAgar or other media for growth of Sclerotinia sclerotiorum, in order todetermine if they are infested with the fungus. Since infection ofcanola with the fungus occurs only by infested petals, this test revealsif and to what extent the fungus is present in the field. Other than thepresence of the fungus, favorable environmental conditions and thepresence of susceptible host are critical for disease development. Plotswere irrigated using a mist irrigation system. Rating was performed byusing two separate parameters, disease incidence and disease severity.Disease incidence is a percentage of plants infected. Disease severityis rated only on infected plants and it implies the extent to whichplants are damaged by the fungal infection and potential yield loss.

In the field, the infection occurred at a late growth stage when plantsare physiologically more tolerant to Sclerotinia. Therefore, there areno data on early infection. Disease severity is rated on the scale 1 to9, where 1 is a dead, broken off plant and 9 is a plant with no symptomsof disease.

SCLEROTINIA RATING SCALE

1—Prematurely ripened or dead plant

3—Large lesion>30mm, weak and girdled stem

5—Large lesion>30mm, stiff and nearly girdled stem

7—Small lesion<30mm, stiff and not girdled stem

9—No symptoms

Intermediate scores can be assigned if symptom severity falls in betweendefined scores.

The results in Table 6 compare the greenhouse results with the fieldresults.

TABLE 6 Field reaction of the oxalate oxidase transgenic lines toSclerotinia at Stirk's compared to indoor reaction to Sclerotinia andthe level of enzyme activity Transgenic Field Field Field Enzyme IndoorEntries and Disease Disease Disease Southern Activity Screening parentalIncidence Severity 1 Severity 2 Analyses T2 Lesion line (%) (1-9) (1-9)(# of copies) OD 550 nm length (mm) 170B3 21.3 7.5+ 6.5+ 2 1.32 95.3164B1 31.9 7.0+ 5.0 1 1.31 80.9 170I2 35.7 6.3 4.8 1 1.22 102.1 164C140.1 6.0 4.5 2 N/A N/A 170J1 46.0 5.5 4.3 2 0.97 98.0 NS1565 51.3 5.33.5 0 0.00 116.3

Table 6, shows a significant decrease in disease severity and incidence,especially ifn the field, that can be attributed to the oxalateoxidase's activity. The level of the enzyme activity is well correlatedwith disease incidence and severity.

The conclusion is that the oxalate oxidase gene in canola is efficientagainst Scierotinia in the field, when compared against thenon-transformed line NS1565. The transformed lines, especially thosehaving a higher level of enzyme activity, exhibited a significantdecrease in disease incidence and severity in the field when compared tothe non-transformed line.

Combining Conventionally Tolerant Canola Lines with Oxalate OxidaseExpressors

In order to identify conventionally tolerant canola lines the stemisolation method of inoculating Sclerotinia was used. The fungus wasgrown on Potato Dextrose Agar (PDA). Agar plugs were cut (approximately4-6mm in diameter) using a cork borer. Plugs were taken from the outsidering of mycelium, before the colony reached the edge of the plate.Canola plants were inoculated at the onset of flowering to midflowering. Stem inoculation was performed by attaching the plug to thestem (mycelial side on the stem) in a leaf axil approximately 10-12 cmabove the soil level. Plants were incubated in the humidity chamberuntil symptoms started developing. Inoculum was then removed and theincubation in humidity was ceased when the lesion length on most of theplants of a susceptible check was at least 10-20mm. Plants were moved tothe greenhouse bench and lesion length was recorded. Lesion lengthdevelopment was observed after 10 days.

The conventionally tolerant line 96SN20002 is of canola quality andoriginates from of a recurrent selection program aimed at enhancedtolerance to Sclerotinia. Tolerant sources of canola quality that wereused to establish the population were originated from the non-canolaquality Asiatic germplasm lines: GP193 and JAP. The selection 96SN20002was made by a single plant selections at Cycle 0-S2 generation (after S0and S1 single plant selections) using the stem inoculation method.Sclerotinia tolerant F4 plants of 96SN20002 were crossed withSclerotinia tolerant transgenic plants of 164B1 in order to generate theF1s for the Scierotinia screening.

The plasmid pPHP7746 was used to transform NS1565 to create 164B1.NS1565 is a spring Brassica napus Pioneer variety which is registered as46A65 in Western Canada. It is susceptible to Sclerotinia.

The experiment was arranged as RCBD (randomised complete block design)with two replications and 4 plants per replication. Plants wereinoculated using the stem inoculation method with the isolate SS4. Planttissue was sampled to estimate enzyme activity at the same time.

A summary of the final lesion length rating and the oxalate oxidaseactivity can be found in Table 7.

TABLE 7 Summary of the final lesion length rating (millimeters) andoxalate oxidase activity of F1 TRANSGENIC/CONVENTIONAL Material,TOLERANT LINES and SUSCEPTIBLE CHECKS. OX-OX Disease Lesion ActivitySeverity Length OD Entries Entry Name (1-9) Mm (Stem) @550 nm * 1Westar - susceptible 2.2 101 0.1 2 Quantum - susceptible 1.2 98 0.1 3NS1565 - susceptible 1.6 103 0.1 4 F5 of 96SN20002 5.4 31 0.2(conventionally tolerant) 5 164B1 2.0 90 1.7 6 QUANTUM/96SN20002 3.5 570.2 7 QUANTUM/164B1 1.9 116 1.0 8 NS1565/96SN20002 3.6 53 0.1 9NS1565/164B1 3.5 62 0.9 10  164B1/QUANTUM 1.7 90 1.0 11  164B1/NS15652.2 102 0.9 12  96SN20002/QUANTUM 3.7 45 0.1 13  96SN20002/NS1565 4.9 480.2 14  96SN2002/164B1 8.6 4 1.0 15  164B1/96SN20002 7.5 12 1.4 * Assayactivity does not have background readings subtracted from values.(0.1˜background in this case)

The test was severe but it revealed the strength of conferred tolerancein the F1s between 164B1 and 96SN20002 and vice versa. This unusualreaction of elevated tolerance of the F1 is based on the interaction ofdifferent tolerances. It also appears that mechanisms of tolerance thatare involved in this synergistic interaction are different. This levelof tolerance was not observed in past greenhouse screening with eitherthe conventional or transgenic material. It is important to note thereare no canola quality lines with tolerance to Sclerotinia presentlyavailable on the market.

The plasmid pPHP8188 was used to transform NS1565 to create the singlecopy transgenic line 156A. Sixty four independent pPHP8188 transformantswere produced. Seventeen were single copy integration events. Transgenicprogeny (T1) was selfed and (T2) homozygous seed identified for theseventeen lines via herbicide spraying. Oxalate oxidase assays wereperformed on the initial transgenics and again on several T2 plants fromseven single copy lines which showed good expression at the T0generation.

An indoor screening experiment using Scierotinia was arranged as RCBD(randomised complete block design) with two replications and 4 plantsper replication. Plants were inoculated using the stem inoculationmethod with the isolate SS4. Plant tissue was sampled to estimate enzymeactivity at the same time.

A summary of the final lesion length rating and the oxalate oxidaseactivity can be found in Table 8.

TABLE 8 pPHP8188 transgenic and control Sclerotinia screening results.Disease Severity OX-OX Activity Vector DS2 (Stem) Scale Lesion LengthLL2 OD reading Entries (if applicable) 1-9 (Stem) mm 550 nm 185TpPHP8188 1.5 129 0.06 WESTAR non trans* 1.6 116 0.07 NS1565-2 non trans*1.5 117 0.08 185U pPHP8188 1.7 115 0.11 186C pPHP8188 2.2 97 0.13NS1565-1 non trans* 2.3 103 0.15 96SN20139 non trans convent 4.2 45 0.15source tolerance* 153D pPHP8188 4.6 51 0.46 185O pPHP8188 4.5 65 0.47153B pPHP8188 3 86 0.48 185Q pPHP8188 2.7 93 0.49 155F pPHP8188 4.7 550.5 154A pPHP8188 1.5 112 0.53 185W pPHP8188 2.1 119 0.59 158F pPHP81883.2 87 0.61 158D pPHP8188 2.5 91 0.72 185I pPHP8188 3.8 80 0.84 F196SN20002/ convent source/ 6.7 22 1.09 164B1 pPHP7746 F1 164B1/pPHP7746/ 6 37 1.2 96SN20002 convent source 156A pPHP8188 3.6 82 1.52164B1 pPHP7746 4 63 1.8 170B3 pPHP7746 5.5 48 1.9 *non-transgenic line.

Line 156A had the highest enzyme expression of the pPHP8188 lines butwas still lower than the best pPHP7746 expressing lines 170B3 and 164B1.In the stem inoculation a correlation of 0.62 for diseaseseverity/lesion length with enzyme activity is seen and indicates thetransgenics exhibit tolerance to Sclerotinia. The test appears to becapable of generally separating very low enzyme activity vs. mid tohigh. Discrepancies are probably due to the low level of toleranceexhibited indoors indicating tests could not detect fine differences.Hybrid combinations were the strongest in terms of tolerance toSclerotinia and the most consistent. Although, differences between theFls (oxalate oxidase expressors and conventional) were not that great asin the pPHP7746 transgenics, it should be noted this is an interactionbetween plants and the fungus, and a pattern was observed.

Efficacy of Oxalate Oxidase Transgenic Canola Against Blackleg andAlternaria

Transgenic canola plants containing the oxalate oxidase gene were testedfor resistance to two canola pathogens, Blackleg (Phoma lingam) andAlternaria. Disease severity and lesion length were similar for allplants regardless of oxalate oxidase activity. Therefore, expression ofoxalate oxidase alone in canola does not confer resistance to Blackleg(Phoma lingam) or Alternaria.

Induction of Host Defenses in Oxalate Oxidase Transgenic Canola

Transgenic canola plants expressing oxalate oxidase were tested for thepresence of PR-1 protein, chitinase and glucanase by Western blotanalysis as was done in sunflower plants. No expression of PR-1,chitinase or glucanase was found in transgenic canola plants expressingoxalate oxidase.

Conclusion

Sunflower and canola differ as to induction of the host defense systems,but are similar in their significant increase in resistance toScierotinia when a transgenic oxalate oxidase expressing plant iscrossed into a conventionally tolerant background. The synergisticeffect created by oxalate oxidase expression in a tolerant backgroundholds true regardless of the species of plant. Both canola and sunfloweroxalate oxidase expressing plants in a tolerant background havesurprising levels of resistance to Sclerotinia and little to none of thedisease lesion mimic phenotype.

EXAMPLE 3 SOYBEAN

Soybean transgenics were produced by cocultivation of soybeancotyledonary node with Agrobacterium. This was done in accordance withthe protocol defined by U.S. Pat. No. 5,563,055, and hereby incorporatedby reference. The A. tumefaciens strain LBA4404 harboring binary plasmidp11144 which has supermas::oxalate oxidase and a histone promoter fromArabidopsis (2XH4) driving NPT was used to transform the soybeancotyledonary node. After culture on kanamycin containing media toprovide a selective advantage to the transformed cells plants wereregenerated. The resulting plants were screened by oxalate oxidaseenzyme assay. Several plants with oxalate oxidase activity were obtainedfrom each of three elite Pioneer soybean varieties (9151,92B52 and9341). The activity is present in both leaf and stem tissue. The plantsare characterized, enzyme rate are developed and PR protein inductionestimates are done. Each event is selfed and homozygous lines aredeveloped. Some crossing to Scerlotinia resistant and sensitive lines isalso done. Baseline Sclerotinia scores for each of the soybean genotypesare performed and each transgenic is evaluated for resistance toSclerotinia.

EXAMPLE 4 MAIZE Maize Transformation

Maize transformation was accomplished by particle bombardment ofimmature embryos with DNA encoding the oxalate oxidase gene fused to themaize ubiquitin promoter (ubi) and potato proteinase inhibitor II(PINII) 3′ region. Ears PHN46 (U.S. Pat. No. 5,567,861, filed Aug. 3,1993) from both greenhouse and field nursery sources were surfacesterilized in 25% commercial bleach solution containing 0.5% Microdetergent (Micro, International Products Corp., Burlington, N.J.) for 20min. then rinsed twice in sterile distilled water. Immature embryos wereexcised from these ears and placed embryo axis side down on 604 medium(0.4×N6 basal salts, 0.6×N6 macronutrients, 16.6 mM KNO₃, 20 μM AgNO₃,0.6×B5H minor salts, 0.6×B5H Na/Fe EDTA, 0.4×Eriksson's vitamins,0.6×S&H vitamins, 0.5 μM Thiamine HCL, 17.2 mM L-proline, 0.03% caseinhydrolysate, 2% sucrose, 0.06% glucose, 0.2% gelrite, 0.8 mg/l 2,4-D,1.2 mg/l dicamba) 4-5 days prior to particle bombardment. Four hoursprior to bombardment the embryos were transferred with the sameorientation to 604S medium (604 medium with sucrose adjusted to 12%).Tungsten particles were prepared by first cleaning the particles bysuspending them in 0.1 M HNO₃ and subjecting them to constant sonicationon ice for 30 min. The tungsten particles are then rinsed with steriledouble distilled H2O 1×, 100% ethanol 1×, then resuspended in steriledouble distilled H₂O prior to aliquoting at 15 mg/ml. Plasmid PHP10963was cut with restriction enzyme to release a DNA fragment which containstwo genes; ubi:: oxalate oxidase::PINII and ubi::moPAT::PINII. Aquantity of this DNA fragment was associated with the particles prior tobombardment by adding components to a microtube in the following volumesand order: 100 μl tungsten particles, 1 μg DNA /10 μl TE buffer, 100 μl2.5 M CaCl₂, 5 μl 0.1 M spermidine. Each component is added to the tubewhile constantly mixing, the final mixture was sonicated briefly, andthen mixed by vortex for an additional 10 min. Tubes were thencentrifuged briefly and supernatants removed from the particles anddiscarded. Particle associated with DNA were then washed with 500 μl100% ethanol, centrifuged for 30 s followed by removal of the wash, andresuspended in 100 μl 100% ethanol. This suspension was used to provide10 μl aliquots which were pipetted on the macrocarriers following abrief sonication and about 2 min prior to bombardment. Particlebombardment was achieved using a Dupont PDS 1000 He particleacceleration device with 650 psi rupture discs. Following bombardment,the embryos remained on 604S medium for 3 d and were then transferred inthe same orientation to 604A medium (604 medium with 3 mg/1bialophos)for 1 wk. After 1 wk on 604A the bombarded embryos are transferred to604J (604A medium lacking proline and casein hydrolysate, and withreduced AgNO₃ to 5 uM) and subcultured every 2 wk.

Oxalate oxidase enzyme assays—Maize

Twelve maize T0 transformed plants representing at least 5 independentmolecular integration events were tested for the presence of functionaloxalate oxidase enzyme by using the same enzyme assay protocol asdescribed for sunflower tissue except that fresh maize leaf tissue wasused in every case. Four maize leaf punches were used per sample and thecolor develoment reaction was scaled up to a larger final volume of 600μl. Results of the oxalate oxidase enzyme assay are shown in Table 9below.

TABLE 9 Oxalate oxidase enzyme assay results from T0 maize transgenicleaf samples with non-transformed maize B73 as negative control andtransgenic sunflower line (193870) as positive control. No. of ColorDevelopment T0 Plant Code Event Samples Call (Ave. OD550) 725611 1 2Positive 0.17 725609 1 2 Positive 0.74 725610 1 2 Positive 0.72 726371 12 Positive 0.70 726372 1 2 Positive 0.46 725605 3 2 Positive 1.44 7256063 2 Positive 1.29 725607 3 2 Positive 1.10 726370 3 2 Positive 1.13726368 7 2 Negative 0.00 719934 10  2 Positive 1.41 719933 22  2Negative 0.01 B73 (negative na 2 Negative 0.00 control) SF (positive na1 Positive 1.74 control)

Positive calli are then regenerated by the following method. Positivelines are transferred to 288J medium (in one liter brought up to volumewith distilled water: 4.3 g MS salts (GIBCO #1117-074), 0 gmyo-inositol, 5 ml MS vitamin stock solution, 0.5 mg zeatin, 60 gsucrose, 3 g Gelrite, 0.5 mg indole acetic acid, 0.1 μM absissic acidplus selective agent if desired to initiate plant regeneration.Following somatic embryo maturation (2-4 weeks), well-developed somaticembryos are transferred to 272V medium (in one liter brought up tovolume with distilled water: 4.3 g of MS salts, 0.1 g myo-inositol, 5mls MS vitamin stock, 40 g sucrose, and 6 g bacto-agar) for germinationand transferred to the lighted culture room. Approximately 7-10 dayslater, developing plantlets are transferred to 272V medium in tubes for7-10 days until plantlets are well established. Plants are thentransferred to inserts in flats (equivalent to 2.5″ pot) containingpotting soil and grown for 1 week in a growth chamber, subsequentlygrown an additional 1-2 weeks in the greenhouse, then transferred toclassic 600 pots (1.6 gallon) and grown to maturity.

Regenerated plants expressing oxalate oxidase are then tested foroxalate oxidase activity as described earlier. Positive plants are nextassayed for PR-1, chitinase, glucanase, 14-3-3 protein, and SA levels asdescribed earlier. Positive T0 and T1 maize plants expressing oxalateoxidase are tested for resistance to stress inducers, such as fungal,viral, and bacterial diseases; environmental stress; and resistance toinsects.

EXAMPLE 5 Maize Transformation with Galactose Oxidase

Maize callus was bombarded, as described above, with a DNA fragmentcontaining the galactose oxidase gene (pPHP12046, ubiquitin::optimizedPAT::PINII/ubiquitin::galactose oxidase::PINII). The galactose oxidasegene isolated from the Fusarium strain NRRL 2903 and cited in McPherson,et al., J of Biol Chem267(12):8146-8152 (1992), and herein incorporatedby reference, was used in the pPHP12046 construct. The resulting calluswas tested for galactose oxidase activity as described below:

1. Disrupt the callus (approximately 20 mg) in 100 mM Na phosphate pH7.0 plus 25 mM N-ethyl maleimide (0.5 ml). The tissue can behomogenized. One way of homogenizing the tissue is the use an apparatusfor tissue preparation as described in U. S. patent application Ser. No.08/713,507, filed on Sep. 13, 1996, and herein incorporate by reference.

2. Add galactose (25 mM), horseradish peroxidase (10 U/ml), Amplex Red™(70 uM−Molecular Probes)−total volume=200 μl.

3. Incubate until color develops, can be 10 minutes to 4 hours, butpreferably about 1 hour.

4. Centrifuge and decant supernatant.

5. Record absorbance at 572 nm.

One skilled in the art will recognize various ways of measuring thefluorescent Resourfin product that are different from the way describedabove, but which still remain within the spirit and scope of theinvention. Also, the concentration of horseradish peroxidase may bevaried from 0.5 about μl/ml to 20 about μl/ml, and the concentration ofAmplex Red™ may vary from about 20 μM to about 200 μM, depending on thedesired reaction time. Reaction volume and galactose concentration mayvary depending again on desired reaction time. Tissue other than callusmay also be used. For example, galactose oxidase activity can bedetected in all plant parts, including but not limited to, roots, stemsleaves, flowers, pollen, and seed. Again, these variations are commonchanges one skilled in the art would make and still remain within thespirit and scope of the invention. Chloroform may also be added afterincubation with galactose, Amplex Red™, and horseradish peroxidase, inorder to stop the reaction. For the example reaction as described above100 μl of chloroform may be added before centrifugation.

The above described assay detects H₂O₂ generated by galactose oxidasethrough a horse radish peroxidase-mediated 1:1 reaction between H₂O₂ andAmplex Red (10-acetyl-3,7-dihydroxyphenoxazine) that generates highlychromophoric and fluorescent resorufin (Zhou, et al., Anal Biochem253:162-168 (1997)). Unfortunately the assay described in the Zhou, etal. article could not be used to measure galactose directly in disruptedcallus because of a reaction between Amplex Red and substancescontaining free sulfydryl groups which results in H₂O₂-independent colordevelopment. This limitation has been overcome by disrupting tissue inthe presence of N-ethyl maleimide eliminating the free sulfydryl groups(Haugaard, et al., Anal Biochem 116:341-343 (1981)). This is the firsttime a galactose oxidase assay has been applied using Amplex Red.Several other chromogens such as o-dianisidine (Tressel et al., MethEnzymol 90:163-171 (1982)) have been employed along with horse radishperoxidase for detecting H₂O₂ generated by oxidase enzymes, but becauseof the high extinction coefficient of resorufin, Amplex Red Assay hasproven to be a much more sensitive chromogen.

The results of the galactose oxidase assay on maize callus can be seenin FIG. 7. Galactose oxidase can be detected in maize callus.

Positive calli are then regenerated by the following method. Positivelines are transferred to 288J medium (in one liter brought up to volumewith distilled water: 4.3 g MS salts (GIBCO #1117-074), 0.1 gmyo-inositol, 5 ml MS vitamin stock solution, 0.5 mg zeatin, 60gsucrose, 3 g Gelrite, 0.5 mg indole acetic acid, 0.1 μM absissic acidplus selective agent if desired) to initiate plant regeneration.Following somatic embryo maturation (2-4 weeks), well-developed somaticembryos are transferred to 272V medium (in one liter brought up tovolume with distilled water: 4.3 g of MS salts, 0.1 g myo-inositol, 5mls MS vitamin stock, 40 g sucrose, and 6 g bacto-agar) for germinationand transferred to the lighted culture room. Approximately 7-10 dayslater, developing plantlets are transferred to 272V medium in tubes for7-10 days until plantlets are well established. Plants are thentransferred to inserts in flats (equivalent to 2.5″ pot) containingpotting soil and grown for 1 week in a growth chamber, subsequentlygrown an additional 1-2 weeks in the greenhouse, then transferred toclassic 600 pots (1.6 gallon) and grown to maturity.

Regenerated plants expressing galactose oxidase are then tested forlevels of galactose oxidase activity as described earlier. Positiveplants are next assayed for PR-1, chitinase, glucanase, 14-3-3 protein,and SA levels as described earlier. Positive T0 and T1 maize plantsexpressing galactose oxidase are tested for resistance to stressinducers, such as fungal, viral, and bacterial diseases; environmentalstress; and resistance to insects.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method of increasing a plant's resistance to anoxalate-secreting pathogen, the method comprising: a. transforming aplant cell with an oxalate decarboxylase gene, wherein said plant cellis characterized by having an oxalate-secreting pathogen tolerantgenetic background; b. culturing said plant cell under plant growingconditions to produce a regenerated plant; and c. expressing the oxalatedecarboxylase gene in the plant, thereby making the plant more resistantto an oxalate-secreting pathogen.
 2. The method of claim 1, wherein theplant is selected from the group consisting of sunflower, canola,alfalfa, soybean, maize, sorghum, wheat, and rice.
 3. The method ofclaim 1, wherein the pathogen is an oxalate-secreting fungal pathogen.4. The method of claim 3, wherein the pathogen is Sclerotinia.
 5. Anoxalate-secreting pathogen resistant plant made by the method ofclaim
 1. 6. The plant of claim 5, wherein the plant is selected from thegroup consisting of sunflower, canola, alfalfa, soybean, maize, sorghum,wheat, and rice.
 7. The plant of claim 5, wherein the pathogen is anoxalate-secreting fungal pathogen.
 8. The plant of claim 7, wherein thepathogen is Sclerotinia.